Fungal Endophytes for Improved Crop Yields and Protection from Pests

ABSTRACT

The invention provides a synthetic combination of a crop and at least one fungal endophyte, wherein the crop is a host plant of the endophyte. Provided are also methods and compositions for producing such synthetic combinations. The endophyte reproduces and enhances the agronomic characteristics of the crop. Methods for inoculating the host plant with the endophyte, for propagating the host-endophyte combination, and for detecting the presence of the endophyte and of its metabolites within a host plant are also described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/535,292 filed Nov. 6, 2014, allowed, which claims priority to U.S.Provisional Patent Application Nos. 61/900,929 and 61/900,935, bothfiled Nov. 6, 2013, which are herein incorporated by reference in theirentirety.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named 32600US_Sequence_Listing.txt, includes 77 sequences and is 33 kilobytes asmeasured in Microsoft Windows operating system and was created on Dec.5, 2015, is filed electronically herewith and incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to fungal endophytes of agricultural cropsfor improving yield and/or for protection from pests.

DESCRIPTION OF RELATED ART

Fungal endophytes are fungi that internally colonize plant tissueswithout causing evident damage or disease. Particular fungal endophytes,such as mycorrhiza, survive within various host plant tissues, oftencolonizing the intercellular spaces of host leaves, stems, flowers orroots. The symbiotic endophyte-host relationships can provide severalfitness benefits to the host plant, such as enhancement of nutrition,and/or increased drought tolerance. Root-colonizing mycorrhizae surviveon photosynthetic carbohydrates from the plant, and in return, aid inthe solubilization and uptake of water and minerals to the host, whichcan lead to the promotion of seed germination and plant growth.Additionally, the association of a fungal endophyte with a host plantcan provide tolerance to a variety of biotic and abiotic stresses. Hostgrowth, fitness promotion and protection are thought to be achievedthrough multiple beneficial properties of the endophyte-hostassociation. For instance, the endophytic organisms may producegrowth-regulating substances to induce biomass production and alkaloidsor other metabolites. Additionally, fungal endophytes may directlysuppress or compete with disease-causing microbes, protecting the plantfrom potential pathogens.

SUMMARY OF THE INVENTION

In one aspect, the invention provides methods for improving a trait inan agricultural plant comprising contacting an agricultural seed of saidplant with a formulation comprising a purified facultative fungalendophytes of at least one species, wherein the endophytes are capableof producing substances that are beneficial to plants or detrimental topests or both, and wherein the endophytes are present in the formulationin an amount effective to modulate the colonization frequencies of theendophytes that are native to the agricultural plant grown from the seedcompared to a reference seed that is planted in an agriculturalenvironment, and to provide a benefit to the seeds or the agriculturalplants grown from the seeds.

In another aspect, the invention provides methods for providing abenefit to an agricultural plant comprising treating said plant, theseed of said plant, or the rhizosphere of said plant or seed with acomposition comprising purified facultative fungal endophytes and anagriculturally-acceptable carrier, wherein the endophyte is capable ofat least one of: reducing pest reproduction, killing pests, anddeterring pests, and wherein the endophyte is present in the compositionin an amount effective to provide a benefit to the seeds or theagricultural plants derived from the seeds.

In yet another aspect, the invention provides methods for providing abenefit to an agricultural plant, comprising obtaining a syntheticcombination of an agricultural plant seed and a purified facultativefungal endophyte, wherein the endophyte is capable of at least one of:reducing pest reproduction, killing pests, and deterring pests, andwherein the endophyte is present in the synthetic combination in anamount effective to provide a benefit to the seeds or the agriculturalplants derived from the seeds.

In another embodiments, methods of producing a plant with anon-naturally occurring ratio of endophytes is provided, where themethods comprise contacting an agricultural seed of the plant with aformulation comprising facultative fungal endophytes of at least onespecies, wherein endophytes are present in the formulation in an amounteffective to modulate the colonization frequencies of the endophytesthat are native to the agricultural plant grown from the seed comparedto a reference seed that is planted in an agricultural environment,wherein the plant with the non-naturally occurring ratio of endophyteshas an improved trait as compared to a plant with a naturally-occurringratio. In a further aspect, the facultative fungal endophytes arecapable of producing substances that are beneficial to plants ordetrimental to pests or both.

In another aspect, the invention provides methods for altering thesystemic defensive pathway in a plant comprising contacting anagricultural seed of said plant with a formulation comprising a purifiedfacultative fungal endophytes of at least one species, wherein theendophytes are capable of producing substances that are beneficial toplants or detrimental to pests or both, and wherein the endophyte ispresent in the synthetic combination in an amount effective to modulatethe level of at least one phytohormone within an agricultural plantgrown from the plant seed, and to provide a benefit to the seeds or theagricultural plants grown from the seeds. In a further aspect, thefacultative fungal endophytes are capable of producing substances thatare beneficial to plants or detrimental to pests or both.

In other embodiments, the invention provides methods of modulating thecolonization frequencies of endophytes that are native to theagricultural plant grown from the seed compared to a reference seed thatis planted in an agricultural environment, comprising contacting theseed of the agricultural plant with a formulation comprising facultativefungal endophytes of at least one species, and wherein endophytes arepresent in the formulation in an amount effective to modulate thecolonization frequencies of native endophytes and to provide a benefitto the seeds or the agricultural plants grown from the seeds. In certainaspects, the native endophytes are of genus Alternaria. In a furtheraspect, the facultative fungal endophytes are capable of producingsubstances that are beneficial to plants or detrimental to pests orboth.

In another aspect, the invention provides methods for altering thesystemic defensive pathway in a plant comprising contacting anagricultural seed of said plant with a formulation comprising a purifiedfacultative fungal endophytes of at least one species, and wherein theendophyte is present in the synthetic combination in an amount effectiveto modulate the level of at least one phytohormone within anagricultural plant grown from the plant seed, and to provide a benefitto the seeds or the agricultural plants grown from the seeds. In afurther aspect, the facultative fungal endophytes are capable ofproducing substances that are beneficial to plants or detrimental topests or both.

In yet another aspect, the invention provides methods of producing aplant with a network of fungal endophytes that comprises endophytes ofthe genus Alternaria, comprising (a) contacting the seed of anagricultural plant with a formulation comprising facultative fungalendophytes of at least one non-Alternaria species, wherein endophytesare present in the formulation in an amount effective to provide abenefit to the seeds or the agricultural plants grown from the seeds,and wherein the plant grown from the seed comprises endophytes of thegenus Alternaria. In a further aspect, the facultative fungal endophytesare capable of producing substances that are beneficial to plants ordetrimental to pests or both.

Also provided herein are synthetic combinations of an agricultural plantseed and a composition comprising purified entomopathogenic fungalendophytes of at least one species, wherein the endophytes are capableof (1) colonizing the agricultural plant grown from the plant seed (2)and at least one of: reducing pest reproduction, killing pests, anddeterring pests, from within the agricultural plant; wherein theendophytes are not of species Beauveria bassiana, and wherein theendophyte is present in the synthetic combination in an amount effectiveto provide a benefit other than enhanced resistance to biotic stress tothe seeds or the agricultural plants derived from the seeds when theseeds or plants are grown in an agricultural setting.

In yet another aspect, the invention provides synthetic combinations ofan agricultural plant seed and a composition comprising purifiedfacultative fungal endophytes of at least one species, wherein theendophyte is present in the synthetic combination in an amount effectiveto modulate the level of at least one phytohormone within anagricultural plant grown from the plant seed, and to provide a benefitto the seeds or the agricultural plants grown from the seeds. In afurther aspect, the facultative fungal endophytes are capable ofproducing substances that are beneficial to plants or detrimental topests or both.

In another embodiment, the invention provides synthetic combinations ofan agricultural plant seed and a composition comprising purifiedfacultative fungal endophytes of at least one species, wherein thefacultative fungal endophytes are present in the synthetic combinationin an amount effective to modulate the colonization frequencies ofendophytes that are native to the agricultural plant grown from the seedcompared to a reference seed that is planted in an agriculturalenvironment, and to provide a benefit to the seeds or the agriculturalplants grown from the seeds. In a further aspect, the facultative fungalendophytes are capable of producing substances that are beneficial toplants or detrimental to pests or both. In certain aspects, thefacultative fungal endophytes are present in the synthetic combinationin an amount effective to modulate the colonization frequencies ofendophytes of genus Alternaria that are native to the agricultural plantgrown from the seed compared to a reference seed that is planted in anagricultural environment.

In a further aspect for certain of these methods and syntheticcombinations, the composition comprising purified facultative fungalendophytes also comprises an agriculturally acceptable carrier.

In a further aspect for certain of these methods and syntheticcombinations, the facultative fungal endophyte may be a filamentousfungal endophyte. In other embodiments, the facultative endophyte may bespore-forming. In yet other embodiments, the facultative fungalendophyte may be a septate fungal endophyte. In yet other embodiments,the facultative fungal endophyte may be a dark septate fungal endophyte.In some embodiments, the facultative endophyte may be an entomopathogen.In some embodiments, the facultative fungal endophyte may belong to thephylum Ascomycota or Basidiomycota. In a further aspect, the facultativefungal endophyte may belong to subphylum Pezizomycotina,Agaricomycotina, or Ustilaginomycotina. In yet another aspect,facultative fungal endophyte may belong to class Sordariomycetes,Dothideomycetes, Agaricomycetes, Ustilaginomycetes, Orbiliomycetes, orEurotiomycetes. In yet another aspect, the facultative fungal endophytemay belong to order Hypocreales, Pleosporales, Capnodiales, Sordariales,Polyporales, Diaporthales, Ustilaginales, Xylariales, Orbiliales,Trichosphaeriales, or Eurotiales.

In a further aspect, the facultative fungal endophyte may be a speciesfrom Table 1, namely Acremonium alternatum, Alternaria alternata,Alternaria brassicae, Alternaria compacta, Alternaria dianthi,Alternaria longipes, Alternaria mali, Alternaria sesami, Alternariasolani, Alternaria sp., Alternaria tenuissima, Ascomycota sp., Bipolarisspicifera, Cercospora canescens, Cercospora capsici, Cercosporakikuchii, Cercospora zinnia, Chaetomium globosum, Chaetomiumpiluliferum, Chaetomium sp., Cladosporium cladosporioides, Cladosporiumsp., Cladosporium uredinicola, Cochliobolus sp, Phanerochaete crassa,Phoma americana, Phoma subherbarum, Phomopsis liquidambari, Phomopsissp., Pleospora sp., Pleosporaceae sp., Polyporales sp., Preussiaafricana, Preussia sp., Pseudozyma sp., Pyrenophora teres,Colletotrichumcapsici, Coniolariella gamsii, Coniothyrium aleuritis,Coniothyrium sp., Corynespora cassiicola, Diaporthe sp., Diatrype sp.,Drechslerella dactyloides, Embellisia indefessa, Epicoccum nigrum,Epicoccum sp., Exserohilum rostratum, Fusarium chlamydosporum, Fusariumsp., Gibellulopsis nigrescens, Gnomoniopsis sp., Lewia infectoria,Mycosphaerella coffeicola, Mycosphaerellaceae sp., Nigrospora oryzae,Nigrospora sp., Nigrospora sphaerica, Paecilomyces sp., Penicilliumcitrinum, Retroconis sp., Rhizopycnis sp., Schizothecium inaequale,Stagonospora sp., Stemphylium lancipes, Thielavia hyrcaniae, Thielaviasp., Ulocladium chartarum, Verticillium sp., Beauveria bassiana,Aspergillus parasiticus, Lecanicillium lecanii, and Paecilomyceslilacinus.

In a further aspect, the facultative fungal endophyte comprises anucleic acid that is at least 97% identical, for example, at least 98%identical, at least 99% identical, at least 99.5% identical, or 100%identical to the nucleic acids provided in any of SEQ ID NO:7 throughSEQ ID NO:77, for example those listed in Example 16.

In another aspect for certain of these methods is an additional step ofpackaging the contacted seeds in a container may be included. In certainaspects, the packaging material may be selected from a bag, box, bin,envelope, carton, or container, and may comprise a dessicant.

In a further aspect for certain of these methods and syntheticcombinations, the benefit to the treated seed or plant grown from thetreated seed is measured at the level of the population, as compared toa reference population of plants. In certain aspects, the facultativefungal endophyte may be providing a benefit to a crop comprising aplurality of agricultural plants produced from the seeds treated withthe endophyte. In certain aspects, the present invention discloses asubstantially uniform population of plants produced by growing thepopulation of seeds described above. In one embodiment, at least 75%, atleast 80%, at least 90%, at least 95% or more of the plants comprise inone or more tissues an effective amount of the endophyte or endophytes.In another embodiment, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%,at least 80%, at least 90%, at least 95% or more of the plants comprisea microbe population that is substantially similar.

In a further aspect for certain of these methods and syntheticcombinations, the plant is grown in an agricultural setting orenvironment, including a greenhouse. In one embodiment, the agriculturalsetting or environment comprises at least 100 plants. In anotherembodiment, the population occupies at least about 100 square feet ofspace, wherein at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% or more than 90% of the population comprises an effective amount ofthe microbe. In another embodiment, the population occupies at leastabout 100 square feet of space, wherein at least about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% or more than 90% of the populationcomprises the microbe in reproductive tissue. In still anotherembodiment, the population occupies at least about 100 square feet ofspace, wherein at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% or more than 90% of the population comprises at least 10 CFUs, 100CFUs, 1,000 CFUs, 10,000 CFUs or more of the facultative fungalendophyte of the invention. In yet another embodiment, the populationoccupies at least about 100 square feet of space, wherein at least about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90% of thepopulation comprises the facultative fungal endophyte of the invention.

In one embodiment, at least 10%, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 75%, at least80%, at least 90%, at least 95% or more of the seeds in the population,contains a viable endophyte or endophytes disposed on the surface of theseeds. In a particular embodiment, at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least75%, at least 80%, at least 90%, at least 95% or more of the seeds inthe population contains at least 10 CFU, for example, at least 30 CFU,at least 100 CFU, at least 300 CFU, at least 1,000 CFU, at least 3,000CFU, at least 10,000 CFU or more, of the endophyte or endophytes coatedonto the surface of the seed.

In a further aspect for certain of these methods and syntheticcombinations, the endophytes that are native to the agricultural plantand whose colonization frequencies or ratios are altered may belong tophylum Ascomycota or Basidiomycota. In yet another aspect, theendophytes that are native to the agricultural plant may be of classLeotiomycetes, Dothideomycetes, Eurotiomycetes, Saccharomycetes,Sordariomycetes, Agaricomycetes, Microbotryomycetes, Tremellomycetes. Inyet another aspect, the native endophytes may belong to orderCapnodiales, Pleosporales, Chaetothythyriales, Eurotiales,Saccharomyceoles, Diaporthales, Hypocreales, Ophiostomatales,Sordariales, Trichosphaeriales, Xylariales, Cantharellales, Corticiales,Polyporales, Russulales, Sporidiobolales, or Tremellales. In a furtheraspect, the native endophytes may belong to genus Davidiellaceae,Mycosphaerellaceae, Pleosporaceae, Didymellaceae, Sporormiaceae,Chaetothyriaceae, Trichocomaceae, Saccharomycetaceae, Gnomoniaceae,Cordycipitaceae, Nectriaceae, Hypocreaceae, Plectosphaerellaceae,Ophiostomataceae, Chaetomiaceae, Lasiosphaeriaceae, Trichosphaeriaceae,Ceraiobasidiaceae, Corticiaceae, Coriolaceae, Peniophoraceae,Sporidiobolaceae, or Tremellaceae. In a further aspect, the endophytesthat are native to the agricultural plant may be a species from Table 2,namely Cladosporium sp., Cladosporium cladosporioides, Davidiella sp.,Cercospora sp., Cercospora beticola, Alternaria sp., Alternariaalternata, Alternaria citri, Alternaria tenuissima, Cochliobolus sp.,Curvularia sp., Exserohilum sp., Lewia sp., Lewia infectoria,Pyrenophora sp., Pyrenophora tritici-repentis, Pleospora sp., Phomaamericana, Preussia africana, Penicillium sp., Thermomyces sp.,Thermomyces lanuginosus, Candida sp., Candida quercitrusa, Candidatropicalis, Cyberlindnera sp., Cyberlindnera jadinii, Kluyveromyces sp.,Kluyveromyces marxianus, Gnomoniopsis sp., Beauveria bassiana, Cordycepssp., Cordyceps bassiana, Fusarium sp., Gibellulopsis nigrescens,Hypocrea sp., Hypocrea lixii, Hypocrea virens, Trichoderma sp.,Trichoderma tomentosum, Verticillium sp., Ophiostoma sp., Ophiostomadendifundum, Chaetomium sp., Chaetomium globosum, Thielavia hyrcaniae,Taifanglania sp., Taifanglania inflata, Schizothecium inaequale,Nigrospora sp., Rhizoctonia sp., Phanerochaete sp, Trametes sp.,Trametes hirsuta, Trametes villose, Rhodotorula sp., Rhodotorulamucilaginosa, Cryptococcus sp. Cryptococcus skinneri, or Tremella sp.

In a further aspect for certain of these methods and syntheticcombinations, the benefit provided by the facultative fungal endophyteto the agricultural plant is an improved agronomic property selectedfrom the group consisting of increased biomass, increased tillering,increased root mass, increased flowering, increased yield, increasedwater use efficiency, reduction of yield loss, altered plant height,decreased time to emergence, increased seedling height, increased rootlength, increased chlorophyll levels, retention of developing flowers,retention of developing fruits, altered phytohormone levels, andenhanced resistance to environmental stress relative to a referenceplant. In some aspects, the benefit provided is the alteration of levelsof at least two phytohormones. In some aspects, the environmental stressis selected from the group consisting of drought stress, cold stress,heat stress, nutrient deficiency, salt toxicity, aluminum toxicity,grazing by herbivores, insect infestation, nematode infection, andfungal infection, bacterial infection and viral infection. In someaspects, the benefit to agricultural plants derived from the seed isincreased yield in a population of said plants by about 5%, 10%, 15%,20%, 30%, 40%, or 45% relative to a reference population of plants. Inother aspects, the benefit to agricultural plants derived from the seedis a reduction of yield loss in a population of said plants by more than40%, 30%, 20%, 10%, 5%, or 1% relative to a reference population ofplants. In some aspects, treatment of seeds with facultative fungalendophytes may decrease thrip damage, decrease fleahopper damage,increase canopy temperature, increase drought tolerance, increase aboveground biomass, and increase below ground biomass in the plants grownfrom the treated seeds.

In a further aspect for certain of these methods and syntheticcombinations, the facultative fungal endophyte is present in thesynthetic combination in an amount effective to obtain at least 50%colonization of the leaves, stems or roots of an agricultural plantgrown from the seed.

In a further aspect for certain of these methods and syntheticcombinations, the facultative fungal endophytes are capable of producingsubstances that are detrimental to pests. In certain aspects, the pestmay be a nematode and/or an insect, for example, a root knot nematode, aaphid, a lygus bug, a stink bug, or combinations thereof.

In a further aspect for certain of these methods and syntheticcombinations, the synthetic combination may comprise at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20facultative fungal endophytes. In one aspect, the invention provides asynthetic combination of a cotton plant or seed and a fungal endophytecomprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 endophytes selected from those in Table 1, whereinthe cotton or seed is a host of the endophyte.

In another aspect, a seed coating is provided comprising a fungalendophyte comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 endophytes from Table 1; and at least onesticker, wherein the fungal endophyte is in contact with the sticker. Incertain aspects, the sticker may comprise, for example, alginic acid,carrageenan, dextrin, dextran, pelgel, polyethelene glycol, polyvinylpyrrolidone, methyl cellulose, polyvinyl alcohol, gelatin, orcombinations thereof. In certain aspects, the sticker may have a weightratio between fungal endophyte and sticker of 1:1-10, 1:10-50, 1:50-100,1:100-500, 1:500-1000, or 1:1000-5000. The seed coating may be a solidor fluid. In certain aspects, the seed coating is a powder. In certainaspects, the fungal endophyte may comprise fungal spores. In variousaspects, the seed coating may comprise about 1, 2, 5, 10, 50, 10², 10³,10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or 10⁹ or more colony forming units per gram orspores per gram.

In certain embodiments, compositions for foliar or soil application maycomprise a fungal endophyte comprising at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 endophytes from Table1, and at least one carrier, surfactant or diluent. In certain aspects,the compositions may comprise may comprise about 1, 2, 5, 10, 50, 10²,10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or 10⁹ or more colony forming units pergram or spores per gram. In various aspects, the composition maycomprise water, a detergent, Triton X, insecticides, fungicides, orcombinations thereof, for example. In further embodiments, seedcompositions comprise a plant seed and the above-described seed coating.In certain aspects, the plant seed comprises a cotton seed, a seed of anagronomically elite plant, a dicot plant seed, and/or a monocot plantseed. In certain aspects, the seed composition may be resistant to apest comprising an insect and/or a nematode.

In yet another aspect, the invention provides methods for preventingpest infestation or increasing yield, which may comprise treating aplant, plant seed, or the rhizosphere of said plant or seed with theendophyte containing compositions described herein. In certain aspects,the method may also comprise identifying a plant or seed as in need ofendophyte treatment. The pest may comprise, for example, a nematodeand/or insect. In certain aspects, the pest may comprise a root knotnematode, a aphid, a lygus bug, a stink bug, or combinations thereof.

In still yet another aspect, methods for preventing pest infestation areprovided comprising obtaining a seed described herein and planting theseed. The method may further comprise identifying a need of preventingpest infestation. In certain aspects, the pest may comprise a nematodeand/or a insect; and/or the pest may comprise a root knot nematode, aaphid, a lygus bug, a stink bug, or combinations thereof.

In a further embodiment, a method for treating a pest infestationcomprises identifying a plant suspected of being infected with a pest,applying an above-described composition to the plant, whereby anendophyte-treated plant is generated. In certain aspects, the pest maycomprise a nematode and/or an insect; and/or the pest may comprise aroot knot nematode, a aphid, a lygus bug, a stink bug, or combinationsthereof

In still yet another aspect, a method of manufacturing pest-resistantseeds is provided comprising providing a fungal endophyte compositioncomprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 endophytes from Table 1, providing seeds; andcombining the seeds with the endophyte composition, wherebypest-resistant seeds are generated. In certain aspects, the methodincreases the percentage of colonization with the endophyte of the plantdeveloping from the seed.

In still yet another aspect, methods of increasing a yield of a crop ora reduction of loss are disclosed comprising providing a fungalendophyte composition comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 endophytes from Table 1; andapplying the endophyte composition to a seed, plant or part thereof,whereby the yield of the crop increases. In certain aspects, the cropmay be cotton, and the increase of yield may be at least about 2%, 3%5%, 15%, 20%, or 25% relative to a crop to which no endophytecomposition has been applied. In certain aspects, the increase of yieldis about 2%-5%, 3%-5%, 5%-10%, 10%-15%, or greater than about 20%, 30%,or more relative to a crop to which no endophyte composition has beenapplied. In certain aspects, the crop is cotton and the increase ofyield comprises reduced boll damage. In certain aspects, the reductionof loss comprises reduction of loss due to insect infestation ordrought, and the loss is less than 50%, 40%, 30%, 20%, 10%, 5%, or 5%relative to a crop to which no endophyte composition has been applied.

Also described herein are commodity plant products comprising a plant orpart of a plant (including a seed) and further comprising thefacultative fungal endophyte described above that is present in adetectable level, for example, as detected by the presence of itsnucleic acid by PCR. In another aspect, disclosed is a method ofproducing a commodity plant product, comprising obtaining a plant orplant tissue from the synthetic combination described above, andproducing the commodity plant product therefrom. The commodity plantproduct can be produced from the seed, or the plant (or a part of theplant) grown from the seed. The commodity plant product can also beproduced from the progeny of such plant or plant part. The commodityplant product can be is selected from the group consisting of grain,flour, starch, seed oil, syrup, meal, flour, oil, film, packaging,nutraceutical product, an animal feed, a fish fodder, a cereal product,a processed human-food product, a sugar or an alcohol and protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: The colonization efficiencies demonstrate that endophytes can bemanipulated in the field. Depicted are the mean+/−SE endophyticcolonization frequencies of cotton seedlings under field conditionsinoculated by seed treatments with different spore concentrations ofeither (left) Paecilomyces lilacinus or (right) Beauveria bassiana.

FIG. 2: The endophytic fungus Paecilomyces lilacinus negatively affectsroot knot nematode (Meloidogyne incognita) reproduction when present asan endophyte in cotton. At high nematode inoculum levels (10,000 eggs),the endophyte reduced egg production in plants following treatment ofseeds with solutions containing either 10⁶ or 10⁷ spores/ml whencompared to untreated control seeds. At field inoculum levels (2000eggs), the presence of the endophyte significantly reduced both gallsand egg production at both seed treatment concentrations.

FIG. 3: Endophytic Chaetomium globosum negatively affects root-knotnematode reproduction. Negative effects of endophytic Chaetomiumglobosum on root-knot nematode gall formation and egg productionfollowing cotton seed soaking treatments in solutions of 0 (untreatedcontrols), 10⁶ and 10⁸ spores/ml. Seedlings were inoculated with 1000nematode eggs and grown in the greenhouse. Egg production by hatchingnematodes that successfully infected the seedlings was quantified 60days later.

FIG. 4A and FIG. 4B: The effect of endophytic fungi on cotton aphids(Aphis gossypii) reproduction. FIG. 4A demonstrates that the presence ofBeauveria bassiana in cotton negatively affects the reproduction ofcotton aphids. FIG. 4B demonstrates that the presence of Paecilomyceslilacinus in cotton negatively affects the reproduction of cottonaphids.

FIG. 5: Effects of Chaetomium globosum on cotton aphids. EndophyticChaetomium globosum in cotton negatively affects cotton aphid populationgrowth rates as evidenced by reduced reproduction after 14 days onendophyte-colonized versus control plants. Cotton plants were grown fromseeds treated by soaking in spore solutions of 0 (control), 10⁶ (low)and 108 (high) spores/ml.

FIG. 6A and FIG. 6B: The effect of the endophytic fungi Beauveriabassiana and Paecilomyces lilacinus on western tarnished plant bugsLygus hesperus (Miridae). FIG. 6A demonstrates that Beauveria bassianaand Paecilomyces lilacinus negatively affect host plant selection ofwestern tarnished plant bugs when present as an endophyte in cotton.FIG. 6B demonstrates that Beauveria bassiana and Paecilomyces lilacinusnegatively affect host plant selection behavior of western tarnishedplant bugs when present as an endophyte in cotton.

FIG. 7A and FIG. 7B: The effect of the endophytic fungi Beauveriabassiana and Paecilomyces lilacinus on southern green stink bugs (Nezaraviridula (Pentatomidae). FIG. 7A demonstrates that Beauveria bassianaand Paecilomyces lilacinus negatively affect host plant selection ofsouthern green stink bugs when present as an endophyte in cotton. FIG.7B demonstrates that Beauveria bassiana and Paecilomyces lilacinusnegatively affect host plant selection behavior of southern green stinkbugs when present as an endophyte in cotton.

FIG. 8: A reduction in cotton boll damage was observed during fieldtrials. Relative to control plants, levels of insect-related boll damagewere lower among plants that were treated by soaking seeds in sporesolutions of Beauveria bassiana and Paecilomyces lilacinus atconcentrations of 10⁶ and 10⁸ spore/ml.

FIG. 9: Foliar application of cotton in the field with spores ofendophytic entomopathogenic fungi improves plant performance. Cotton(variety FM1740B2F) seeds treated with a variety of typical fungicide(Metalaxyl, Triadimenol, Trifloxystrobin, 2-(Thiocyanome-thylthio)benzothioazole) and insecticide (Thiodicarb, Imidacloprid,Chloropyrifos) seed treatments were planted and grown under fieldconditions. The plants were sprayed at the 5th true leaf stage withaqueous solutions of Beauveria bassiana and Paecilomyces fumosoroseus.Sucrose was included (1% wt/vol) as an additional nutritional resourcefor the fungi. Significantly higher first position boll (developingfruit) retention was observed in plants sprayed with Beauveria bassianawithout sucrose and Paecilomyces fumosoroseus plus sucrose.

FIG. 10A and FIG. 10B: Positive effects of fungal endophytes on cottonplant performance under field conditions. FIG. 10A demonstrates an earlyseason trend for higher square retention in the treated versus untreatedplants. FIG. 10B demonstrates that significantly more bolls wereretained in the endophyte treatment groups later in the season, relativeto control. This is demonstrated with both endophyte species used andwith both seed treatment concentration employed (Repeated measuresANOVA: Time, P<0.001; Time*Endophyte, P=0.045, Endophyte, P=0.003).

FIG. 11: Positive effects of fungal endophytes on cotton yields underfield conditions. The data demonstrate that endophyte treatmentsachieved 25% higher yields in treated cotton plants.

FIG. 12A and FIG. 12B: Positive effects of fungal endophytes on sorghum(FIG. 12A) plant height and (FIG. 12B) total fresh biomass under growthchamber seedling assays. Data shown is average plant height (cm) andtotal fresh biomass (g) of n=10 independent replicates. Error barsrepresent ±1 standard error. All three fungal endophytes improve bothtraits relative to the untreated control.

FIG. 13: The in-field modulation of the colonization of endogenouscotton endophytes in (panels A, B) stems and (panels C, D) roots whentreated with fungal endophytes Paecilomyces lilacinus (panels A, C) andBeauveria bassiana (panels B, D). Data shown is a percentage change incolonization relative to the corresponding untreated control and planttissue.

FIG. 14: Average percent difference in yield between endophyte treatedand control cotton plants (n=6 replicate plots in a dryland field,College Station, Tex.) for 15 facultative fungal endophytes in thePhytogen (PHY 499WRF) cultivar.

FIG. 15: Aggregated average percent difference in yield betweenendophyte treated and control cotton plants (n=6 replicate plots in adryland field, College Station, Tex.) for 15 facultative fungalendophytes and two cotton cultivars; Delta Pine (DP 0912B2RF) andPhytogen (PHY 499WRF). Bars represent a 95% confidence interval aroundthe mean.

FIG. 16A and FIG. 16B: Average percent difference in thrip damage (FIG.16A) and fleahopper damage (FIG. 16B) between endophyte treated andcontrol cotton plants. The thrip damage was assessed in the Delta Pine(DP 0912B2RF) cultivar (n=6 replicate plots in a dryland field, CollegeStation, Tex.) for 15 facultative fungal endophytes. 12 out of the 15facultative fungal endophytes tested showed a decrease in thrip damagerelative to the untreated cotton plants. The fleahopper damage wasassessed in cotton plants of the Phytogen (PHY 499WRF) cultivar (n=6replicate plots in a dryland field, College Station, Tex.) for 15facultative fungal endophytes. 6 out of the 15 facultative fungalendophytes tested showed an average decrease in fleahopper damage ascompared to untreated cotton plants.

FIG. 17A and FIG. 17B: Mid-season field-trait measured in June at thedryland trial of (FIG. 17A) root length and (FIG. 17B) belowgroundweight. Data presented is the average of n=10 independent replicates anderror bars represent ±one standard error.

FIG. 18: Mid-season field-trait measured in July at the dryland trial ofcanopy temperature (Celsius) for the (open bars) Delta Pine and (hatchedbars) Phyton cultivars. Data presented is the block-controlled averageof n=10 independent replicates, relative to the control plot and errorbars represent ±one standard error.

FIG. 19: Mid-season field-trait measured in August at the dryland trialof NDVI for the (open bars) Delta Pine and (hatched bars) Phytoncultivars. Data presented is the block-controlled average of n=10independent replicates, relative to the control plot and error barsrepresent ±one standard error.

FIG. 20: Mid-season field-trait measured in August at the dryland trialof first position square retention for the (open bars) Delta Pine and(hatched bars) Phyton cultivars. Data presented is the block-controlledaverage of n=10 independent replicates, relative to the control plot anderror bars represent ±one standard error.

FIG. 21: Mid-season field-trait measured in August at the dryland trialof plant height (cm) for the (open bars) Delta Pine and (hatched bars)Phyton cultivars. Data presented is the block-controlled average of n=10independent replicates, relative to the control plot and error barsrepresent ±one standard error.

FIG. 22: Mid-season field-trait measured in July at the dryland trial ofplant height (cm) for the (open bars) Delta Pine and (hatched bars)Phyton cultivars. Data presented is the block-controlled average of n=10independent replicates, relative to the control plot and error barsrepresent ±one standard error.

FIG. 23: Picture showing increased biomass in the plants treated withendophytes (right half of the image) compared to untreated control (lefthalf of the image).

FIG. 24: Table showing the time to wilt following drought stress in daysfor plants grown from seeds treated with fungal endophytes and control.

FIG. 25: Table showing the time to death following drought stress indays for plants grown from seeds treated with fungal endophytes andcontrol.

DETAILED DESCRIPTION OF THE INVENTION

Endophytic fungi are ubiquitous in nature, infecting virtually allplants in both natural and agronomic ecosystems. Plants commonly harbora diversity of fungi living within their tissues as asymptomaticendophytes that can provide protection from a range of biotic andabiotic stressors. The present disclosure describes certain fungalendophytes that can be pathogens, parasites or antagonists to plantpathogens, insects, and nematode pests, thereby providing health andperformance benefits to crop plants. The symbiotic endophyte-hostrelationships can provide several general health and fitness benefits tothe host plant, such as enhancement of nutrition, increased droughttolerance and/or chemical defense from potential herbivores and oftenenhanced biomass production. Root-colonizing mycorrhizae survive onphotosynthetic carbohydrates from the plant, and in return, aid in thesolubilization and uptake of water and minerals to the host, which canlead to the promotion of seed germination and plant growth.Additionally, the association of a fungal endophyte with a host plantoften provides protection from pathogens or tolerance to a variety ofbiotic and abiotic stresses, such as insect infestation, grazing, wateror nutrient deficiency, heat stress, salt or aluminum toxicity, andfreezing temperatures. Host growth and fitness promotion and protectionare thought to be achieved through multiple beneficial properties of theendophyte-host association.

These fungal endophytes provided in Table 1 were originally collected asfungal endophytes of cotton. These endophytic fungi can be inoculated tolive within cotton using either seed, soil or foliar applications andexhibited surprisingly beneficial effects by providing protection frompest infestation. Pests can be nematode and/or insect pests. Inaddition, these endophytic fungi have an unexpected beneficial effect oncotton yield.

Described is the application of beneficial fungi to establishendophytically within crop plants to improve plant performance and yieldwhile conferring protection against insect and nematode pests. In thisregard, the present invention overcomes the limitations of the prior artsuch as the susceptibility of the fungi to degradation by UV light,desiccation or heat after exposure to the environment followingapplication as an inundative soil or foliar biopesticide. Inoculationand endophytic establishment of the fungi within the plant protects thefungi from UV light, desiccation, and unfavorable temperatures, whileharboring the fungi in the very plant tissues they are intended toprotect. Introducing fungi to live endophytically within plants requiresno genetic modification of the plant or microorganisms, and the fungithemselves can be a source for natural products. In various embodiments,the fungal inoculant can be formulated and applied, for example, astreatment of seeds, in furrow applications, before or during planting,or as foliar application after plant germination, and after inoculation,the fungal endophytes provide season-long protective effects and highercrop yields (approximately 25% higher). In certain embodiments, theincrease of yield is about 5%, 10%, 15%, 20%, 30%, 40%, 45%, 50%, orgreater than 50% relative to a crop to which no endophyte compositionhas been applied. In further embodiments, the increase of yield is theresult of reduction of loss that comprises reduction of loss due toinsect infestation or drought and the loss is less than 50%, 40%, 30%,20%, 10%, 5%, or 5% relative to a crop to which no endophyte compositionhas been applied. In certain embodiments, the crop is cotton and thereduction of loss comprises reduced boll damage.

Thus, in one aspect, the invention provides a combination (also termed a“symbiotum”) of a host plant and an endophyte that allows for improvedagronomic properties of host plants. The combination may be achieved byartificial inoculation, application, or other infection of a host plantor seeds thereof, such as a cotton plant or seed thereof, or host planttissues, with a fungal endophyte strain of the present invention. Thus,a combination achieved by such an inoculation is termed a “synthetic”combination, synthetic composition, synthetic seed coating, and/orsynthetic pest-resistant seed composition. The fungal endophyte may bepresent in intercellular spaces within plant tissue, such as the root.Its presence may also occur or may also be maintained within a plant orplant population by means of grafting or other inoculation methods suchas treating seeds, plants or parts thereof with endophyte mycelia, orendophyte spores. In certain embodiments, the plant, part of the plant,roots, seed, or leaves are sterilized to remove microorganisms beforeapplying the endophyte. In particular embodiments, seeds are sterilizedto remove native endophytes before adding the endophyte compositionsherein described. In certain aspects, the ability of the seed togerminate is not affected by the sterilization.

The invention also provides methods for detecting the presence of thefungal endophyte of the present invention within a host plant. This maybe accomplished, for instance, by isolation of total DNA from tissues ofa potential plant-endophyte combination, followed by PCR, oralternatively, Southern blotting, western blotting, or other methodsknown in the art, to detect the presence of specific nucleic or aminoacid sequences associated with the presence of a fungal endophyte strainof the present invention. Alternatively, biochemical methods such asELISA, HPLC, TLC, or fungal metabolite assays may be utilized todetermine the presence of an endophyte strain of the present inventionin a given sample of crop tissue. Additionally, methods foridentification may include microscopic analysis, such as root staining,or culturing methods, such as grow out tests or other methods known inthe art (Deshmukh et al. 2006). In particular embodiments, the roots ofa potential grass plant-endophyte combination may be stained with fungalspecific stains, such as WGA-Alexa 488, and microscopically assayed todetermine fungal root associates.

In certain embodiments, the agronomic qualities may be selected from thegroup consisting of: increased biomass, increased tillering, increasedroot mass, increased flowering, increased seed yield, and enhancedresistance to biotic and/or abiotic stresses, each of these qualitiesbeing rated in comparison to otherwise identical plants grown under thesame conditions, and differing only with respect to the presence orabsence of a fungal endophyte. The synthetic combinations and methods ofthe present invention may be applied to respond to actual or anticipatedstresses. Such stresses may include, for instance, drought (waterdeficit), cold, heat stress, nutrient deficiency, salt toxicity,aluminum toxicity, grazing by herbivores, insect infestation, nematodeinfection, and fungal, bacteria or viral infection, among others.

The present disclosure provides, in one embodiment, fungal endophytesselected from those in Table 1 that negatively affect the reproductionof insect herbivores feeding on leaves above ground (cotton aphids,Aphis gossypii) and plant parasitic nematodes attacking roots belowground (root knot nematodes, Meloidogyne incognita). In addition,improved plant performance and yields in colonized versus uncolonizedcontrol plants may be observed in field trials employing seed treatmentwith such endophytes. Plant growth enhancement and increased resistanceto root knot nematodes was demonstrated in cotton, for example,employing Chaetomium globosum as an endophyte in greenhouse trials. Inaddition and as a further non-limiting illustrative example, usingBeauveria bassiana as an endophyte in cotton, reductions in insect(cotton aphid) reproduction was demonstrated in both greenhouse andfield trials. The endophytic presence of Paecilomyces lilacinus andBeauveria bassiana also had negative effects on the host selectionbehavior of key sucking bug pests (Lygus hesperus and Nezara viridula)that attack developing flowers and fruits in cotton. Furthermore, infield trials using Beauveria bassiana as an endophyte in cotton positiveeffects on plant performance and higher yields in endophyte colonizedversus uncolonized control plants was demonstrated.

Metabolomic differences between the plants can be detected using methodsknown in the art. For example, a biological sample (whole tissue,exudate, phloem sap, xylem sap, root exudate, etc.) from theendophyte-associated and reference agricultural plants can be analyzedessentially as described in Fiehn et al., (2000) Nature Biotechnol., 18,1157-1161, or Roessner et al., (2001) Plant Cell, 13, 11-29. Suchmetabolomic methods can be used to detect differences in levels inhormones, nutrients, secondary metabolites, root exudates, phloem sapcontent, xylem sap content, heavy metal content, and the like.

In another embodiment, the present invention contemplates methods ofcoating the seed of a plant with a plurality of endophytes, as well asseed compositions comprising a plurality of endophytes on and/or in theseed. The methods according to this embodiment can be performed in amanner similar to those described herein for single endophyte coating.In one example, multiple endophytes can be prepared in a singlepreparation that is coated onto the seed. The endophytes can be from acommon origin (i.e., a same plant). Alternatively, the endophytes can befrom different plants.

Where multiple endophytes are coated onto the seed, any or all of theendophytes may be capable of conferring a beneficial trait onto the hostplant. In some cases, all of the endophytes are capable of conferring abeneficial trait onto the host plant. The trait conferred by each of theendophytes may be the same (e.g., both improve the host plant'stolerance to a particular biotic stress), or may be distinct (e.g., oneimproves the host plant's tolerance to drought, while another improvesphosphate utilization). In other cases the conferred trait may be theresult of interactions between the endophytes.

DEFINITIONS

In the description and tables herein, a number of terms are used. Inorder to provide a clear and consistent understanding of thespecification and claims, the following definitions are provided. Unlessotherwise noted, terms are to be understood according to conventionalusage by those of ordinary skill in the relevant art.

When a term is provided in the singular, the inventors also contemplateaspects of the invention described by the plural of that term. Thesingular form “a,” “an,” and “the” include plural references unless thecontext clearly dictates otherwise. For example, the term “a cell”includes one or more cells, including mixtures thereof.

The term “comprising” is intended to mean that the compositions andmethods include the recited elements, but not excluding others.“Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. Thus, a composition consistingessentially of the elements as defined herein would not exclude tracecontaminants from the isolation and purification method andagriculturally acceptable carriers. “Consisting of” shall mean excludingmore than trace elements of other ingredients and substantial methodsteps for applying the compositions of this invention. Embodimentsdefined by each of these transition terms are within the scope of thisinvention.

Biological control: the term “biological control” and its abbreviatedform “biocontrol,” as used herein, is defined as control of a pest,pathogen, or insect or any other undesirable organism by the use of atleast one endophyte.

A “composition” is intended to mean a combination of active agent and atleast another compound, carrier or composition, inert (for example, adetectable agent or label or liquid carrier) or active, such as apesticide.

As used herein, an “agricultural seed” is a seed used to grow plants inagriculture (an “agricultural plant”). The seed may be of a monocot ordicot plant, and is planted for the production of an agriculturalproduct, for example grain, food, fiber, etc. As used herein, anagricultural seed is a seed that is prepared for planting, for example,in farms for growing. Agricultural seeds are distinguished fromcommodity seeds in that the former is not used to generate products, forexample commodity plant products.

As used herein, a “commodity plant product” refers to any composition orproduct that is comprised of material derived from a plant, seed, plantcell, or plant part of the present invention. Commodity plant productsmay be sold to consumers and can be viable or nonviable. Nonviablecommodity products include but are not limited to nonviable seeds andgrains; processed seeds, seed parts, and plant parts; dehydrated planttissue, frozen plant tissue, and processed plant tissue; seeds and plantparts processed for animal feed for terrestrial and/or aquatic animalconsumption, oil, meal, flour, flakes, bran, fiber, and any other foodfor human or animal consumption; and biomasses and fuel products. Anysuch commodity plant product that is derived from the plants of thepresent invention may contain at least a detectable amount of thespecific and unique DNA corresponding to the endophytes describedherein. Any standard method of detection for polynucleotide moleculesmay be used, including methods of detection disclosed herein.

As used herein, the phrase “agronomically elite plants” refers to agenotype or cultivar with a phenotype adapted for commercialcultivation. Traits comprised by an agronomically elite plant mayinclude biomass, carbohydrate, and/or seed yield; biotic or abioticstress resistance, including drought resistance, insect resistance,fungus resistance, virus resistance, bacteria resistance, coldtolerance, and salt tolerance; improved standability, enhanced nutrientuse efficiency, and reduced lignin content.

In certain embodiments, cotton agronomically elite plants include, forexample, known cotton varieties AM 1550 B2RF, NG 1511 B2RF, NG 1511B2RF, FM 1845LLB2, FM 1944GLB2, FM 1740B2F, PHY 499 WRF, PHY 375 WRF,PHY 367 WRF, PHY 339 WRF, PHY 575 WRF, DP 1252 B2RF, DP 1050 B2RF, DP1137 B2RF, DP 1048 B2RF, and/or DP 1137 B2RF.

As used herein, the phrase “culture filtrate” refers to broth or mediaobtained from cultures inoculated with a strain of fungi and allowed togrow. The media is typically filtered to remove any suspended cells,leaving the nutrients, hormones, or other chemicals.

As used herein, the term “endophyte” refers to an organism capable ofliving within a plant or plant tissue. An endophyte may comprise afungal organism that may confer an increase in yield, biomass,resistance, or fitness in its host plant. Fungal endophytes may occupythe intracellular or extracellular spaces of plant tissue, including theleaves, stems, flowers, or roots.

The phrase “pest resistance” refers to inhibiting or reducing attackfrom pests. Pest resistance provides at least some increase in pestresistance over that which is already possessed by the plant.

As used herein, the term “genotypes” refers to the genetic constitutionof a cell or organism.

As used herein, the term “phenotype” refers to the detectablecharacteristics of a cell or organism, which characteristics are eitherthe direct or indirect manifestation of gene expression.

As used herein, the phrase “host plant” refers to any plant that anendophytic fungi colonizes. In certain embodiments, the host plantcomprises progeny of colonized plant.

As used herein, the phrase “increased yield” refers to an increase inbiomass or seed weight, seed or fruit size, seed number per plant, seednumber per unit area, bushels per acre, tons per acre, kilo per hectare,carbohydrate yield, or cotton yield. Such increased yield is relative toa plant or crop that has not been inoculated with the endophyte. Incertain embodiments, the increase yield is relative to other commonlyused pest treatments or other methods of addressing the biotic orabiotic stress.

As used herein, the phrase “biomass” means the total mass or weight(fresh or dry), at a given time, of a plant tissue, plant tissues, anentire plant, or population of plants, usually given as weight per unitarea. The term may also refer to all the plants or species in thecommunity (community biomass).

As used herein, “sticker” refers to compounds to enhance binding ofspores to the seed surface. Non-limiting examples of such compounds arealginic acid, carrageenan, dextrin, dextran, pelgel, polyetheleneglycol, polyvinyl pyrrolidone, methyl cellulose, polyvinyl alcohol, orgelatin.

As used herein, an “agriculturally acceptable” excipient or carrier isone that is suitable for use in agriculture without undue adverse sideeffects to the plants, the environment, or to humans or animals whoconsume the resulting agricultural products derived therefromcommensurate with a reasonable benefit/risk ratio.

As used herein, the term “synthetic” or the phrase “syntheticcombination” refers to an artificial combination that includes myceliaand/or spores of a endophyte that is or leads to an endophyticfungal-host relationship (also termed a “symbiotum”) of a host plant andan endophyte. The synthetic combination may be achieved, for example, byartificial inoculation, application, or other infection of a host plant,host plant seeds, or host plant tissues with the endophyte. In addition,the combination of host plant and an endophyte may be achieved byinoculating the soil or growth media of the plant.

The present invention contemplates the use of “isolated” microbe. Asused herein, an isolated microbe is a microbe that is isolated from itsnative environment, and carries with it an inference that the isolationwas carried out by the hand of man. An isolated microbe is one that hasbeen separated from at least some of the components with which it waspreviously associated (whether in nature or in an experimental setting)or occurs at a higher concentration, viability, or other functionalaspect than occurring in its native environment. Therefore, an“isolated” microbe is partially or completely separated from any othersubstance(s) as it is found in nature or as it is cultured, propagated,stored or subsisted in naturally or non-naturally occurringenvironments. Specific examples of isolated microbes include partiallypure microbes, substantially pure microbes and microbes cultured in amedium that is non-naturally occurring.

As used herein, a microbe is considered to be “native” to a plant or aportion of the plant, and is said to be “natively” present in the plantor a portion of plant, if that plant or portion of the plant containsthe microbe, for example, in the absence of any contacting with themicrobe preparation, or contains the microbe at much lowerconcentrations than the contacting with the microbe preparation wouldprovide.

Some of the methods described herein allow the colonization of plantseeds by microbes. As used herein, a microbe is said to “colonize” aplant or seed when it can exist in a symbiotic or non-detrimentalrelationship with the plant in the plant environment, for example on, inclose proximity to or inside a plant, including the seed.

A “population” of plants, as used herein, refers to a plurality ofplants that were either grown from the seeds treated with the endophytesas described herein, or are progeny of a plant or group of plants thatwere subjected to the inoculation methods. The plants within apopulation are typically of the same species, and/or typically share acommon genetic derivation.

EXAMPLES Example 1 Creating Spore Suspensions and Treatment of Seeds

Cultivation of plants and endophytic fungi strains: The cotton seedvariety used in particular embodiments was variety LA122 (available fromAll-Tex Seed, Inc., Levelland, Tex. 79336). Paecilomyces lilacinus andChaetomium globosum were obtained from cotton plants as described(Ek-Ramos et al. 2013, PLoS ONE 8(6): e66049.doi:10.1371/journal.pone.0066049). Persons of ordinary skill in the artcan obtain endophytes suitable for performing the various embodiments ofthe present invention by performing the procedures described therein. Inshort, plant samples were rinsed in tap water and surface sterilized byimmersion in 70% ethanol for 5 min, 10% bleach solution for 3 min, andrinsed twice with autoclaved distilled water. Samples were blotted dryusing autoclaved paper towels. Five individual surface sterilizedleaves, squares and bolls (N=15 total samples) were randomly selectedand imprinted onto fresh potato dextrose agar (PDA) and V8 media as away to monitor surface sterilization efficiency. For endophyteisolation, leaves were cut in small fragments of approximately 1 cm².Squares and bolls were cut in six pieces. Any fiber present was removedand cut into six smaller pieces. Leaf fragments were placed upside downon PDA and V8 medium plates in triplicate. Each plate contained 3 leaffragments for a total of 9 fragments assayed per plant. For squarescollected early in the season, 3 slices per square were plated on PDAand V8 media as with the leaf fragments. Because of similarity in sizeand location within a plant, when collected later in the season, squaresand bolls from a given plant were plated together on petri dishescontaining two square slices, two boll slices and two pieces of fiber.Antibiotics Penicillin G (100 Units/mL) and Streptomycin (100 μg/mL)(Sigma, St Louis, Mo., USA) were added to the media to suppressbacterial growth. All plates were incubated in the dark at roomtemperature for, in average, two weeks until growth of fungal endophytehyphae from plant tissues was detected.

An inclusive combination of morphological and molecular fungal endophyteidentification was employed for identification. Once fungal hyphae weredetected growing from the plant material, samples were taken to obtainpure fungal isolates. For identification by PCR, genomic DNA wasextracted from mycelium of each isolated fungal strain, following achloroform:isoamyl alcohol 24:1 protocol and fungal specific primerswere used to amplify the ITS (Internal Transcribed Spacer) region ofnuclear ribosomal DNA. This region is the primary barcoding marker forfungi and includes the ITS1 and ITS2 regions, separated by the 5.8Sribosomal gene. In order to avoid introducing biases during PCR(taxonomy bias and introduction of mismatches), it has been suggested toamplify the ITS1 region only, therefore the primers ITS1 (5′ TCC GTA GGTGAA CCT GCG G 3′) (SEQ ID NO:5) and ITS2 (5′ GCT GCG TTC TTC ATC GAT GC3′) (SEQ ID NO:6) were used to amplify and sequence the ˜240 by ITS1region of each one of the isolated fungal strains. The resultingsequences were aligned as query sequences with the publicly availabledatabases GenBank nucleotide, UNITE and PlutoF. The last two arespecifically compiled and used for fungi identification. Table 1provides a list of endophytes identified and useful in the presentinvention. All of these endophytes belong to phylum Ascomycota,subphylum Pezizomycotina, except for Phanerochaete crassa, which belongsto phylum Basidiomycota, subphylum Agaricomycotina, and Pseudozyma sp,which belongs to phylum Basidiomycota, subphylum Ustilaginomycotina.Table 1 shows the species/genus, family, order, subclass, class, and theSEQ ID NO corresponding to the ˜240 bp ITS1 region for each one of theisolated fungal strains, except for Beauveria bassiana, Aspergillusparasiticus, Lecanicillium lecanii, and Paecilomyces lilacinus, wherethe sequences shown includes the ITS1, ITS2, 5.8S, 18S, and 28Ssequences and were obtained from the UNITE database for GenBank numbersJF837090, JX857815, FJ643076, and EU553283, respectively.

TABLE 1 endophytes identified and useful in the present inventionGenus/Species Family Order Subclass Class SEQ ID NO. AcremoniumIncertaesedis Hypocreales Hypocreomycetidae Sordariomycetes 7 alternatumAlternaria Pleosporaceae Pleosporales Pleosporomycetidae Dothideomycetes8 alternata Alternaria Pleosporaceae Pleosporales PleosporomycetidaeDothideomycetes 9 brassicae Alternaria Pleosporaceae PleosporalesPleosporomycetidae Dothideomycetes 10 compacta Alternaria dianthiPleosporaceae Pleosporales Pleosporomycetidae Dothideomycetes 11Alternaria Pleosporaceae Pleosporales Pleosporomycetidae Dothideomycetes12 longipes Alternaria mali Pleosporaceae PleosporalesPleosporomycetidae Dothideomycetes 13 Alternaria sesami PleosporaceaePleosporales Pleosporomycetidae Dothideomycetes 14 Alternaria solaniPleosporaceae Pleosporales Pleosporomycetidae Dothideomycetes 15Alternaria sp. Pleosporaceae Pleosporales PleosporomycetidaeDothideomycetes 16 Alternaria Pleosporaceae PleosporalesPleosporomycetidae Dothideomycetes 17 tenuissima Bipolaris PleosporaceaePleosporales Pleosporomycetidae Dothideomycetes 18 spicifera CercosporaMycosphaerellaceae Capnodiales Dothideomycetidae Dothideomycetes 19canescens Cercospora Mycosphaerellaceae Capnodiales DothideomycetidaeDothideomycetes 20 capsici Cercospora Mycosphaerellaceae CapnodialesDothideomycetidae Dothideomycetes 21 kikuchii CercosporaMycosphaerellaceae Capnodiales Dothideomycetidae Dothideomycetes 22zinnia Chaetomium Chaetomiaceae Sordariales SordariomycetidaeSordariomycetes 23 globosum Chaetomium Chaetomiaceae SordarialesSordariomycetidae Sordariomycetes 24 piluliferum Chaetomium sp.Chaetomiaceae Sordariales Sordariomycetidae Sordariomycetes 25Cladosporium Cladosporiaceae Capnodiales DothideomycetidaeDothideomycetes 26 cladosporioides Cladosporium sp. CladosporiaceaeCapnodiales Dothideomycetidae Dothideomycetes 27 CladosporiumCladosporiaceae Capnodiales Dothideomycetidae Dothideomycetes 28uredinicola Cochliobolus sp Pleosporaceae PleosporalesPleosporomycetidae Dothideomycetes 29 Phanerochaete PhanerochaetaceaePolyporales Incertae sedis Agaricomycetes 30 crassa Phoma Incertae sedisPleosporales Pleosporomycetidae Dothideomycetes 31 americana PhomaIncertae sedis Pleosporales Pleosporomycetidae Dothideomycetes 32subherbarum Phomopsis Diaporthaceae Diaporthales SordariomycetidaeSordariomycetes 33 liquidambari Phomopsis sp. Diaporthaceae DiaporthalesSordariomycetidae Sordariomycetes 34 Pleospora sp. PleosporaceaePleosporales Pleosporomycetidae Dothideomycetes 35 Pleosporaceae sp.Pleosporaceae Pleosporales Pleosporomycetidae Dothideomycetes 36Preussia africana Sporormiaceae Pleosporales PleosporomycetidaeDothideomycetes 37 Preussia sp. Sporormiaceae PleosporalesPleosporomycetidae Dothideomycetes 38 Pseudozyma sp. UstilaginaceaeUstilaginales Ustilaginomycetidae Ustilaginomycetes 39 PyrenophoraPleosporaceae Pleosporales Pleosporomycetidae Dothideomycetes 40 teresColletotrichum Glomerellaceae Incertae sedis SordariomycetidaeSordariomycetes 41 capsici Coniolariella Incertae sedis XylarialesXylariomycetidae Sordariomycetes 42 gamsii Coniothyrium ConiothyriaceaePleosporales Pleosporomycetidae Dothideomycetes 43 aleuritisConiothyrium sp. Coniothyriaceae Pleosporales PleosporomycetidaeDothideomycetes 44 Corynespora Corynesporascaceae PleosporalesPleosporomycetidae Dothideomycetes 45 cassiicola Diaporthe sp.Diaporthaceae Diaporthales Sordariomycetidae Sordariomycetes 46 Diatrypesp. Diatrypaceae Xylariales Xylariomycetidae Sordariomycetes 47Drechslerella Orbiliaceae Orbiliales Orbiliomycetidae Orbiliomycetes 48dactyloides Embellisia Pleosporaceae Pleosporales PleosporomycetidaeDothideomycetes 49 indefessa Epicoccum Pleosporaceae PleosporalesPleosporomycetidae Dothideomycetes 50 nigrum Epicoccum sp. PleosporaceaePleosporales Pleosporomycetidae Dothideomycetes 51 ExserohilumPleosporaceae Pleosporales Pleosporomycetidae Dothideomycetes 52rostratum Fusarium Nectriaceae Hypocreales HypocreomycetidaeSordariomycetes 53 chlamydosporum Fusarium sp. Nectriaceae HypocrealesHypocreomycetidae Sordariomycetes 54 Gibellulopsis PlectosphaerellaceaeIncertae sedis Hypocreomycetidae Sordariomycetes 55 nigrescensGnomoniopsis sp. Glomerellaceae Incertae sedis HypocreomycetidaeSordariomycetes 56 Lewia infectoria Pleosporaceae PleosporalesPleosporomycetidae Dothideomycetes 57 Mycosphaerella MycosphaerellaceaeCapnodiales Dothideomycetidae Dothideomycetes 58 coffeicolaMycosphaerellaceae Mycosphaerellaceae Capnodiales DothideomycetidaeDothideomycetes 59 sp. Nigrospora Incertae sedis TrichosphaerialesIncertae sedis Sordariomycetes 60 oryzae Nigrospora sp. Incertae sedisTrichosphaeriales Incertae sedis Sordariomycetes 61 Nigrospora Incertaesedis Trichosphaeriales Incertae sedis Sordariomycetes 62 sphaericaPaecilomyces sp. Trichocomaceae Eurotiales EurotiomycetidaeEurotiomycetes 63 Penicillium Trichocomaceae Eurotiales EurotiomycetidaeEurotiomycetes 64 citrinum Retroconis sp. Incertae sedis Incertae sedisIncertae sedis Incertae sedis 65 Rhizopycnis sp. Incertae sedis Incertaesedis Incertae sedis Dothideomycetes 66 Schizothecium LasiosphaeriaceaeSordariales Sordariomycetidae Sordariomycetes 67 inaequale Stagonosporasp. Phaeosphaeriaceae Pleosporales Pleosporomycetidae Dothideomycetes 68Stemphylium Pleosporaceae Pleosporales PleosporomycetidaeDothideomycetes 69 lancipes Thielavia Chaetomiaceae SordarialesSordariomycetidae Sordariomycetes 70 hyrcaniae Thielavia sp.Chaetomiaceae Sordariales Sordariomycetidae Sordariomycetes 71Ulocladium Pleosporaceae Pleosporales Pleosporomycetidae Dothideomycetes72 chartarum Verticillium sp. Plectosphaerellaceae Incertae sedisHypocreomycetidae Sordariomycetes 73 Beauveria CordycipitaceaeHypocreales Hypocreomycetidae Sordariomycetes 74 bassiana AspergillusTrichocomaceae Eurotiales Eurotiomycetidae Eurotiomycetes 75 parasiticusLecanicillium Cordycipitaceae Hypocreales HypocreomycetidaeSordariomycetes 76 lecanii Paecilomyces Trichocomaceae EurotialesEurotiomycetidae Eurotiomycetes 77 lilacinus

TABLE 1 List of endophytes:

Acremonium alternatum, Alternaria alternata, Alternaria brassicae,Alternaria compacta, Alternaria dianthi, Alternaria longipes, Alternariamali, Alternaria sesami, Alternaria solani, Alternaria sp., Alternariatenuissima, Ascomycota sp., Bipolaris spicifera, Cercospora canescens,Cercospora capsici, Cercospora kikuchii, Cercospora zinnia, Chaetomiumglobosum, Chaetomium piluliferum, Chaetomium sp., Cladosporiumcladosporioides, Cladosporium sp., Cladosporium uredinicola,Cochliobolus sp, Phanerochaete crassa, Phoma americana, Phomasubherbarum, Phomopsis liquidambari, Phomopsis sp., Pleospora sp.,Pleosporaceae sp., Polyporales sp., Preussia africana, Preussia sp.,Pseudozyma sp., Pyrenophora teres, Colletotrichumcapsici, Coniolariellagamsii, Coniothyrium aleuritis, Coniothyrium sp., Corynesporacassiicola, Diaporthe sp., Diatrype sp., Drechslerella dactyloides,Embellisia indefessa, Epicoccum nigrum, Epicoccum sp., Exserohilumrostratum, Fusarium chlamydosporum, Fusarium sp., Gibellulopsisnigrescens, Gnomoniopsis sp., Lewia infectoria, Mycosphaerellacoffeicola, Mycosphaerellaceae sp., Nigrospora oryzae, Nigrospora sp.,Nigrospora sphaerica, Paecilomyces sp., Penicillium citrinum, Retroconissp., Rhizopycnis sp., Schizothecium inaequale, Stagonospora sp.,Stemphylium lancipes, Thielavia hyrcaniae, Thielavia sp., Ulocladiumchartarum, Verticillium sp., Beauveria bassiana, Aspergillusparasiticus, Lecanicillium lecanii, Paecilomyces lilacinus.

Beauveria bassiana was cultured from a commercially obtained strain(available from Botanigard). Beauveria bassiana, Paecilomyces lilacinus,and Chaetomium globosum were cultured on potato dextrose agar media(PDA). Stock spore concentration solutions of each fungi were made byadding 10 ml of sterile water to the fungi plates and scraping them freeof the agar with a sterile scalpel. The resulting mycelia and sporesobtained were then filtered into a sterile beaker utilizing a cheesecloth to filter out the mycelia, thereby creating stock solutions. Ahaemocytometer was used to measure and calculate spore concentrations ofthe stock solutions. The desired concentrations were created bydilution, and seeds were placed into spore suspensions with the desiredspore concentrations. In various embodiments, the final treatmentconcentrations can be about 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or 10⁹spores/ml which can be reached by serial dilutions in sterile water orin an appropriate solution or buffer.

For seed inoculation, the seeds were surface sterilized prior to soakingthem in spore suspensions with the desired concentration by immersionthe seeds in 70% ethanol for 3 minutes with constant shaking followed byincubation in 2% NaOCl for 3 minutes; followed by three washes insterile water. The third sterile water wash was plated onto potatodextrose agar media (PDA) to confirm that surface sterilization waseffective. Seeds were then soaked for 24 hours in beakers containingspore suspensions with two different concentrations of fungi. Controlgroup seeds were treated with sterile water only. Spore concentrationsfor Beauveria bassiana were zero (control), 1×10⁶ (treatment 1) and1×10⁹ (treatment 2) and for Paecilomyces lilacinus or Chaetomiumglobosum were zero (control), 1×10⁶ (treatment 1) and 1×10⁷ (treatment2). These beakers were incubated for 24 hours at 32° C. in a culturechamber until next day for planting (24 hr).

Soaked seeds were planted in L22 mix soil (Borlaug Institute, TexasA&M). All plants were grown in a laboratory greenhouse at ˜28° C. with anatural light photoperiod. There was no fertilization of the plants, andwatering was done consistently across all treatments as needed.

Direct seed inoculation: In particular embodiments, individual seeds andthe surrounding soil can be directly inoculated with the spore solution(10²-10³, 10³-10⁴, 10⁴-10⁵, 10⁶-10⁷, or 10⁷-10⁸ spores/ml) at plantingbefore covering the seed with soil.

In various embodiments, any seed or plant treatments that are suitablefor application of biological agents to seeds or plants and known topersons having ordinary skill in the art can be employed.

Example 2 Application of Endophyte Spores as a Dry Powder Composition

In addition to application of a spore solution for seed treatment, theendophytes or endophyte spores can also be applied as dry powder orusing a sicker such as methyl cellulose for seed treatment. In certainembodiments, the concentration may be at least 10⁵, 10⁶, 10⁷, 10⁸, 10⁹,or higher colony forming units or spores/g dry weight.

In certain embodiments, endophytes can be grown in fungi cultivationmedia in a fermenter. Endophytic mycelial fragments or spores can becollected, dried and ground. A sticker such as carboxymethyl cellulosemay also be added to the ground endophytic material.

In certain embodiments the weight ratio between endophytic material andsticker may be between 1:10-50, 1:50-100, 1:100-500, or 1:500-1000 toobtain the seed coating or seed inoculation material. This seedinoculation material can be applied to seeds. In various embodiments,the weight ratio between seed inoculation material and seed may be1:10-50, 1:50-100, 1:100-500, 1:500-1000, or 1:1000-5000.

Example 3 Soil (in Furrow) Endophyte Treatments

Soil drench (in furrow) application may be performed by applying anendophyte composition to the surface of the soil and/or seed duringplanting. In particular embodiments, the endophyte composition maycomprise an endophyte suspension or an endophyte dry powder formulation.In various embodiments the endophyte may comprise mycelia and/or spores.In particular embodiments, the soil drench application may compriseapplying the endophyte composition to the surface of the soil directlyabove each seed. In certain embodiments, the endophyte composition maycomprise 0.01-0.1, 0.1-1, or 1-10 ml endophyte suspension, which may bea endophyte spore suspension.

Soil inoculation: In certain embodiments, seeds can be planted intoinoculated soil. The inoculum can be obtained by multiplying theendophyte on fungal growth media. The fungal growth media can be potatodextrose agar media (PDA). In other embodiments the fungal growth mediacan be as wheat grain. In a non-limiting example, 100 g of wheat graincan be washed and soaked overnight in sterile water. Excess water can bedrained, seeds dried on paper towel, packed in a 500 ml conical flaskand autoclaved at 15 psi for 1 h. One milliliter of the endophyticfungal spore suspension (10⁷ spores/ml) can be inoculated to the flask,and the cultures can be incubated at 25° C. for 2 weeks. To avoidclumping, the flasks can be shaken vigorously to separate the grain andbreak the mycelial mat. Approximately 5 g of inoculum can be placed insoil at planting. In certain embodiments, the inoculum can be placed inthe soil at the same time or within 1 month of planting the seeds. Incertain embodiments, the seeds may comprise sterilized seeds.

Example 4 Foliar Endophyte Treatments

Plants were inoculated via foliar application at the third true leafstage by spraying the surface of fully expanded leaves to run-off with aspore suspension (10⁸ spores/ml) using a hand-held plastic sprayer (1L). In certain embodiments, endophyte spore suspensions were made inwater. In certain embodiments, the water was supplemented with adetergent. In a particular non-limiting example, the spore suspensioncontained 0.02% Triton X 100 as a detergent.

Foliar endophyte treatment may be performed using any suitable methodknown to a person having ordinary skill in the art. In particular,foliar endophyte treatment may be performed using a sprayer by directlyspraying leaves with an endophyte suspension, which may be a endophytespore suspension.

FIG. 9 demonstrates that foliar application of cotton in the field withspores of endophytic entomopathogenic fungi improved plant performance.Cotton (variety FM1740B2F) seeds were treated with a variety of typicalfungicide (Metalaxyl, Triadimenol, Trifloxystrobin,2-(Thiocyanome-thylthio) benzothioazole) and insecticide (Thiodicarb,Imidacloprid, Chloropyrifos), and seed treatments were planted and grownunder field conditions. The plants were sprayed at the 5th true leafstage with aqueous solutions of Beauveria bassiana and Paecilomycesfumosoroseus. Sucrose was included (1% wt/vol) as an additionalnutritional resource for the fungi. Significantly higher first positionboll (developing fruit) retention was observed in plants sprayed withBeauveria bassiana without sucrose and P. fumosoroseus plus sucrose.

Example 5 Confirmation of Plant Colonization by Endophytic Fungi

Plants were individually placed in plastic bags, which were labeled withplant number, treatment, and final aphid number, and stored in 4° C.until the next day for endophyte confirmation. Half of each plant wasutilized for plating on PDA agar and the other half was freeze-dried forto conduct diagnostic PCR assays for endophyte confirmation. The surfacesterilization protocol and plating of third sterile water wash on PDA totest for surface contamination was conducted as described above. Fordiagnostic PCR assays, plant tissue was freeze-dried and DNA wasextracted utilizing the CTAB protocol (Doyle & Doyle, 1987,Phytochemistry Bulletin 19:11-15). The oligonucleotide primer sequencessynthesized were based upon a NCBI BLAST search corresponding to thelaboratory culture sequence results isolated (Ek-Ramos et al., 2013).Sense and antisense oligonucleotide sequences for Beauveria bassianawere: 5′-CGGCGGACTCGCCCCAGCCCG-3′ (SEQ ID NO:1) and5′-CCGCGTCGGGGTTCCGGTGCG-3′ (SEQ ID NO:2) respectively. Theoligonucleotides used to amplify Paecelomyces lilacinus were: 5′CTCAGTTGCCTCGGCGGGAA 3′ (SEQ ID NO:3) and 5′ GTGCAACTCAGAGAAGAAATTCCG 3′(SEQ ID NO:4).

The PCR protocol consisted of a denaturation step at 95° C. for 5 min,followed by alignment of oligonucleotides at 56° C. for 2 min and anextension step of 7 min at 72° C. with a total of 35 cycles. The PCRproducts were visualized in a 2% agarose gel containing 1% ethidiumbromide. Electrophoresis was performed at 70 volts for 30 min.

Example 6 Endophytic Fungi can be Manipulated in the Field

A field trial using isolates of Paecilomyces lilacinus and Beauveriabassiana was conducted during the summer. A randomized block design withfive replicate plots that were planted with seeds that were inoculatedby soaking for 9 hr in three different aqueous spore concentrations (0,10⁶, or 10⁸ spores/ml) of the candidate endophyte (such as Paecilomyceslilacinus or Beauveria bassiana). Each plot consisted of four 15.24 m(40 ft) rows, each separated by 101.6 cm (40 in).

Colonization efficiency: At the first true leaf stage, four plants fromeach plot for a total of 20 plants per treatment were randomly sampledand tested for colonization by each of the candidate endophytes.Colonization frequencies were determined by incubating surfacesterilized root, stem and leaf fragments on PDA media and observing forfungal growth. Colonization frequencies are reported as the number ofplants per treatment group with at least one positively colonized plantfragment.

The high endophytic colonization frequency of seedlings by Paecilomyceslilacinus or Beauveria bassiana demonstrates that the presence ofspecific endophytes can be manipulated under field planting conditions(FIG. 1).

Example 7 Cotton Aphid Reproduction Test

A colony of A. gossypii was reared on cotton in cages in a greenhousekept at approximately 28° C. with natural light photoperiod. Secondinstar nymphs were placed directly onto endophyte-treated cotton plantsand control plants. Ten plants were utilized per treatment group and tenaphids were placed per plant. After plants were inoculated with theaphids, the plants were placed in individual plastic 45×20 cm cups andsealed with no-see-um mesh (Eastex products, NJ) to avoid aphid movementfrom plant to plant. In one embodiment, the plants used were 13 daysold, approximately in the first true leaf stage, and aphids were left toreproduce for seven days under greenhouse conditions. In anotherembodiment, aphids were left to reproduce for 14 days on plantsinitially 20 days old at the beginning of the experiment, approximatelyin the third true leaf stage. At the end of each embodiment, aphidnumbers were counted and recorded per individual plant. The presence ofBeauveria bassiana or Paecilomyces lilacinus as an endophyte in cottonsignificantly reduced the reproduction of cotton aphids on endophytetreated plants versus untreated control plants (FIG. 4A, 4B, and FIG. 5)

Example 8 Fungal Endophytes Reduce Nematode Reproduction

Plants were germinated from treated and untreated control seeds in anenvironment chamber and then transplanted to soil in pots 11 days afterplanting. Two replicate seedlings per treatment were sampled to examinethe endophyte colonization efficiency by surface sterilization andplating on PDA agar. Nematode treatment group seedlings were treatedwith either 2,000 or 10,000 eggs/plant at day six after transplanting.Plants were harvested and processed 6 weeks after nematode inoculation.The numbers of galls per gram of root tissue and total egg numbers inthe population for each plant were quantified to compare nematodeperformance between endophyte-treated and untreated (control) plants.

FIGS. 2 and 3 demonstrate that the endophytic fungi Paecilomyceslilacinus and Chaetomium globosum negatively affected root knot nematode(Meloidogyne incognita) reproduction when present as an endophyte incotton. At high nematode inoculum levels (10,000 eggs), Paecilomyceslilacinus reduced egg production in plants following treatment of seedswith solutions containing either 10⁶ or 10⁷ spores/ml when compared tountreated control seeds. At field inoculum levels (2000 eggs), thepresence of Paecilomyces lilacinus significantly reduced both galls andegg production at both seed treatment concentrations. EndophyticChaetomium globosum negatively affects root-knot nematode reproduction.Negative effects of endophytic Chaetomium globosum on root-knot nematodegall formation and egg production were demonstrated following cottonseed soaking treatments in solutions of 0 (untreated controls), 10⁶ and10⁸ spores/ml.

Example 9 Effect of Fungal Endophytes on Insects

Endophyte-treated and control plants were grown from non-transgeniccotton seeds (Gossypium hirsutum) (variety LA122, AllTex Seed Co.).Seeds were soaked for 24 hours in beakers containing 10⁸ spores/mlsolutions of the fungi utilized plus sterile water-only as a control.The beakers were placed in a 32° C. culture chamber overnight (approx. 9h) until planting the next day. The plants were grown under bothgreenhouse and field conditions. Greenhouse plants were first germinatedin seedling trays and then transferred to 30 cm pots. Field grown plantswere concurrently planted and grown.

Behavioral assays: No-choice and choice behavioral assays were conductedto compare the response of western tarnished plant bugs (L. hesperus)and green stink bugs (N. viridula) to squares and bolls fromendophyte-treated and untreated plants. The assays were conducted at 30°C. in 10 cm diameter petri dishes with a thin layer of 2% agar on thebottom to provide moisture for the squares (L. hesperus assays) andbolls (N. viridula assays) from experimental plants offered to theinsects during the observations. For no-choice assays, a single squareor boll was inserted by the base into the agar in the center of thedish. A single young adult (1-7 days post molt) insect was placed ineach dish and covered with the top. A total of 30 insects were observedin each trial with N=10 insects each in the Beauveria bassiana,Paecilomyces lilacinus and control treatment groups. The L. hesperusno-choice trials were replicated four times (N=40 per treatment) withsquares from greenhouse grown plants used in all but one trial. The N.viridula no-choice trials were replicated three times (N=20 pertreatment) with bolls from greenhouse grown plants used in one trial.

Choice tests were conducted under the similar conditions using the samearenas, but with two equal sized squares (L. hesperus) or bolls (N.viridula) placed 4 cm apart in the center of the petri dish. The twosquares or bolls per arena were from an untreated control plant andeither a Beauveria bassiana or Paecilomyces lilacinus treated plant. Atotal of 20 insects were observed in each trial, with N=10 each in theBeauveria bassiana vs. control and Paecilomyces lilacinus vs. controltreatment groups. The L. hesperus and N. viridula choice trials wereboth replicated twice (N=20 per treatment) with squares from field-grownplants in all trials.

Insects were observed for 6 hours per trial using a point samplingprocedure for both the no-choice and choice assays. Preliminaryobservations indicated that the insects of both species were more activeat the beginning of the assay, thus staged sampling schedule was adoptedwith observations recorded at 5 minute intervals early in the assay(0-60 min), 15 minute intervals in the middle (61-180 min) and 30 minuteintervals late (181-360 min) in the assay. At each sampling interval,the insects were recorded as either off the square/boll or feeding orroosting upon the square/boll.

Data analysis: In the no-choice assays, the proportion of insectsobserved either feeding or resting upon cotton squares (L. hesperus) orbolls (N. viridula) was compared between treatment groups at eachobservation point across the duration of the assay using the WilcoxonSigned Ranks Test. To test for variation in responses over time, foreach individual the proportion of observations either feeding or uponthe plant sample was calculated for early (0-60 min), middle (61-180min) and late (181-360 min) periods of the assay and compared acrosstreatment groups using a repeated measures analysis of variance (ANOVA)with the endophyte treatment group as the main factor and time as therepeat effect. The observed frequency of individuals failing to makecontact with squares or bolls from endophyte-treated plants was comparedto the expected frequency of individuals failing to do so based on thecontrol group using a X2 test. Among the insects that did make contactwith either a square or boll, the time to first contact (latency) wascompared among treatment groups using a one-way ANOVA. All analysesincluding tests of normality and homogeneity of variances were conductedin SPSS 21 (SPSS Inc.).

Results of the L. hesperus no-choice assays: Over the duration of theassay, a significantly higher proportion of L. hesperus individuals overtime was observed in contact with and feeding upon squares fromuntreated control plants relative to those from either of the Beauveriabassiana or Paecilomyces lilacinus endophyte treatment groups (WilcoxonSigned Ranks test, P<0.0001 for both comparisons) (FIG. 6A). Repeatedmeasures ANOVA indicated a significant effect of time (F_(1,116)=86.175;P<0.001) with a higher proportion of insects contacting the square asthe assay progressed (FIG. 6B). There was also a significant effect ofendophyte treatment (F_(2,116)=4.929; P=0.009) with no significant timeX endophyte treatment interaction (F_(2,116)=1.015; P=0.366). Of the 40insects in each treatment group, 12.5% of the control group failed tomake contact with the square over the course of the assay, while asignificantly higher 35% and 32.5% the Beauveria bassiana andPaecilomyces lilacinus treatment group insect respectively failed tomake contact (X2 test, P<0.0001). Among the insects that did makecontact with a square, there was significant difference in the latencyto first contact among the treatment groups (F_(2.85)=7.225; P<0.0001)with the control group exhibiting a shorter latency to contact thaneither the Beauveria bassiana (posthoc LSD test; P=0.001) orPaecilomyces lilacinus endophyte treatment groups (posthoc LSD test;P=0.006 (FIG. 6A).

Results of the L. hesperus choice assays: In simultaneous choice tests,L. hesperus individuals selected squares from untreated control plantsmore often than those from endophyte-treated plants. Response ratioswere significantly greater than 0.5 over the duration of the assays,indicating that the insects non-randomly selected bolls from controlplants over bolls from plants endophytically colonized by either (A)Beauveria bassiana (P<0.0001; Wilcoxon Signed Ranks test) or (B)Paecilomyces lilacinus (P<0.0001; Wilcoxon Signed Ranks test)(FIG. 6B).

Results of the N. viridula no-choice assays: Over the duration of theassay, a significantly higher proportion of N. viridula individuals overtime was observed in contact with and feeding upon bolls from untreatedcontrol plants relative to those from either of the Beauveria bassianaor Paecilomyces lilacinus endophyte treatment groups (Wilcoxon SignedRanks test, P<0.0001 for both comparisons)(FIG. 7A). Repeated measuresANOVA indicated a significant effect of time (F_(1,116)=86.175; P<0.001)with a higher proportion of insects contacting the square as the assayprogressed (FIG. 1), There was also a significant effect of endophytetreatment (F_(2,116)=4.929; P=0.009) with no significant time Xendophyte treatment interaction (F_(2,116)=1.015; P=0.366). Of the 40insects in each treatment group, 12.5% of the control group failed tomake contact with the square over the course of the assay, while asignificantly higher 35% and 32.5% the Beauveria bassiana andPaecilomyces lilacinus treatment group insect respectively failed tomake contact (X2 test, P<0.0001). Among the insects that did makecontact with a square, there was significant difference in the latencyto first contact among the treatment groups (F_(2.85)=7.225; P<0.0001)with the control group exhibiting a shorter latency to contact thaneither the Beauveria bassiana (posthoc LSD test; P=0.001) orPaecilomyces lilacinus endophyte treatment groups (posthoc LSD test;P=0.006 (FIG. 7B).

Example 10 More Bolls are Retained after Endophyte Treatment

During the field trial, cotton phenology and development was quantifiedusing a plant mapping and information system developed specifically forcotton to track fruit development and retention by the plant as a meansof monitoring plant development and stress (COTMAN™, Cotton Inc.). Onemeasure of cotton stress is the retention of developing flowers(squares) and fruits (bolls) in the first fruiting position on branches.First position squares and bolls were measured on 5 plants per row intwo rows in each of the five replicate plots (N=10 plants per plot) foreach treatment group.

FIG. 10 demonstrates that early in the growing season as flowers beginto develop, a trend for higher square retention in the endophyte-treatedplants relative to controls was observed. This trend continued later inthe season as evidenced by significantly higher boll retention among theendophyte treatment groups relative to the untreated control plants.

FIG. 8 demonstrates reduction in cotton boll damage during field trials.Relative to control plants, levels of insect-related boll damage werelower among plants that were treated by soaking seeds in spore solutionsof Beauveria bassiana and Paecilomyces lilacinus at concentrations of10⁶ and 10⁸ spore/ml. Positive effects of fungal endophytes on cottonplant performance under field conditions.

Example 11 Endophyte Treatment Increases Yield

At the end of the field trial employing endophyte treatment andtreatment plants, plots were machine harvested with a 1-row picker.Surprisingly, the final yields at harvest were significantly higher thanexpected (25% higher than the untreated controls). Unexpectedly,treatment with Paecilomyces lilacinus or Beauveria bassiana resulted inhigher yields than untreated control plants with regardless of theinitial seed treatment concentration. (FIG. 11)

Example 12 Endophyte Treatment of Sorghum Increased Growth in theGreenhouse

The effect of the described microbial compositions on sorghum was testedin a seedling assay. Sorghum bicolor seeds were surface sterilized usingethanol and bleach as described in Example 1 for cotton. Three strains(B. bassiana, P. fumosoroseus, and P. lilacinus) were prepared asconidia suspensions at 10⁷ conidia/ml, and coated on the sorghum seedsas described in Example 1. Control seeds were soaked in sterile waterinstead of a conidia suspension. Planted seeds were held in constantgrowth chamber conditions for two weeks at a replication of 10. At theend of two weeks, the plants were removed from the growth chamber andthe plant height and biomass were measured. FIG. 12A shows the increasein plant height when applied with the described microbial compositionrelative to the control (p<0.05). FIG. 12B shows the increase in plantbiomass in plants grown from seed that were treated with the describedmicrobial composition relative to the control (p<0.05).

Example 13 Treatment with Fungal Endophytes Modulates the ColonizationFrequencies of Native Endophytes

To determine whether endophyte seed treatments could alter themicrobiome of the plant grown from the seed, cotton seeds were treatedwith spore suspensions of Paecilomyces lilacinus or Beauveria bassiana.Plants were grown in the field as part of a field trial planted andmaintained under standard agricultural practices. Endophytic fungi wereisolated on PDA media separately from surface-sterilized above-groundstem/leaf and below-ground root tissue to assess changes in themicrobial community. The comparison shown in FIG. 13 is relative to thefungal endophyte communities in untreated control plants. The resultsshow that these treatments can alter the colonization rates of nativefungal endophytes.

Fungal endophyte treatments may alter the colonization frequencies ofany of the fungal endophytes naturally present in plants. To determinewhat other native endophytes may be affected by seed treatments withfungal endophytes, the identity of cotton fungal endophytes isolatedfrom plants of two commercial cotton varieties, CG3787B2RF andPHY499WRF, were assessed. The samples were obtained during a varietytrial near Lubbock, Tex., USA identified as Lubbock-RACE. One singlehealthy leaf was collected from each of nine individual plants sampledper variety across multiple replicate plots arranged in a randomizedblock design to control for spatial variation in the field. To identifythe fungal endophyte species, whole genomic DNA was extracted and theribosomal DNA internal transcribed spacer (ITS) region was amplified asa barcode for 454 pyrosequencing using ITS1F forward and ITS2 reverseuniversal fusion primers. The fungal endophytes identified in thisexperiment, along with those shown in FIG. 13, are listed in Table 2.

TABLE 2 Native fungal endophytes that may be altered by seed treatmentswith other fungal endophytes Phylum Class Order Family Genus speciesAscomycota Leotiomycetes Leotiomycetes Geomyces auratus DothideomycetesBotryosphaeriales Botryosphaeriaceae Macrophomina sp. DothideomycetesCapnodiales Davidiellaceae Dothideomycetes Capnodiales DavidiellaceaeCladosporium sp. Dothideomycetes Capnodiales Davidiellaceae Cladosporiumcladosporioides Dothideomycetes Capnodiales Davidiellaceae Davidiellasp. Dothideomycetes Capnodiales Mycosphaerellaceae Cercospora sp.Dothideomycetes Capnodiales Mycosphaerellaceae Cercospora beticolaDothideomycetes Pleosporales Dothideomycetes Pleosporales PleosporaceaeDothideomycetes Pleosporales Pleosporaceae Alternaria sp.Dothideomycetes Pleosporales Pleosporaceae Alternaria alternataDothideomycetes Pleosporales Pleosporaceae Alternaria citriDothideomycetes Pleosporales Pleosporaceae Alternaria porriDothideomycetes Pleosporales Pleosporaceae Alternaria tenuissimaDothideomycetes Pleosporales Pleosporaceae Cochliobolus sp.Dothideomycetes Pleosporales Pleosporaceae Curvularia sp.Dothideomycetes Pleosporales Pleosporaceae Epicoccum sp. DothideomycetesPleosporales Pleosporaceae Exserohilum sp. Dothideomycetes PleosporalesPleosporaceae Lewia sp. Dothideomycetes Pleosporales Pleosporaceae Lewiainfectoria Dothideomycetes Pleosporales Pleosporaceae Pyrenophora sp.Dothideomycetes Pleosporales Pleosporaceae Pyrenophora triticirepentisDothideomycetes Pleosporales Pleosporaceae Pleospora sp. DothideomycetesPleosporales Didymellaceae Phoma americana Dothideomycetes PleosporalesSporormiaceae Preussia africana Eurotiomycetes ChaetothyrialesEurotiomycetes Chaetothyriales Chaetothyriaceae EurotiomycetesEurotiales Trichocomaceae Eurotiomycetes Eurotiales TrichocomaceaeAspergillus sp. Eurotiomycetes Eurotiales Trichocomaceae Penicillium sp.Eurotiomycetes Eurotiales Trichocomaceae Thermomyces sp. EurotiomycetesEurotiales Trichocomaceae Thermomyces lanuginosus SaccharomycetesSaccharomycetales Saccharomycetes Saccharomycetales SaccharomycetaceaeSaccharomycetes Saccharomycetales Saccharomycetaceae Candida sp.Saccharomycetes Saccharomycetales Saccharomycetaceae Candida quercitrusaSaccharomycetes Saccharomycetales Saccharomycetaceae Candida tropicalisSaccharomycetes Saccharomycetales Saccharomycetaceae Cyberlindnera sp.Saccharomycetes Saccharomycetales Saccharomycetaceae Cyberlindnerajadinii Saccharomycetes Saccharomycetales SaccharomycetaceaeKluyveromyces sp. Saccharomycetes Saccharomycetales SaccharomycetaceaeKluyveromyces marxianus Sordariomycetes Sordariomycetes DiaporthalesGnomoniaceae Gnomoniopsis sp. Sordariomycetes HypocrealesCordycipitaceae Beauveria bassiana Sordariomycetes HypocrealesCordycipitaceae Cordyceps sp. Sordariomycetes HypocrealesCordycipitaceae Cordyceps bassiana Sordariomycetes HypocrealesNectriaceae Sordariomycetes Hypocreales Nectriaceae Fusarium sp.Sordariomycetes Hypocreales Hypocreaceae Sordariomycetes HypocrealesHypocreaceae Gibellulopsis nigrescens Sordariomycetes HypocrealesHypocreaceae Hypocrea sp. Sordariomycetes Hypocreales HypocreaceaeHypocrea lixii Sordariomycetes Hypocreales Hypocreaceae Hypocrea virensSordariomycetes Hypocreales Hypocreaceae Trichoderma sp. SordariomycetesHypocreales Hypocreaceae Trichoderma tomentosum SordariomycetesHypocreales Plectosphaerellaceae Verticillium sp. SordariomycetesOphiostomatales Ophiostomataceae Sordariomycetes OphiostomatalesOphiostomataceae Ophiostoma sp. Sordariomycetes OphiostomatalesOphiostomataceae Ophiostoma dendifundum Sordariomycetes SordarialesChaetomiaceae Chaetomium sp. Sordariomycetes Sordariales ChaetomiaceaeChaetomium globosum Sordariomycetes Sordariales Chaetomiaceae Thielaviahyrcaniae Sordariomycetes Sordariales Chaetomiaceae Taifanglania sp.Sordariomycetes Sordariales Chaetomiaceae Taifanglania inflataSordariomycetes Sordariales Lasiosphaeriaceae Schizothecium inaequaleSordariomycetes Trichosphaeriales Trichosphaeriaceae Nigrospora sp.Sordariomycetes Xylariales Amphisphaeriaceae Truncatella angustataBasidiomycota Agaricomycetes Cantharellales CeratobasidiaceaeRhizoctonia sp. Agaricomycetes Corticiales Corticiaceae AgaricomycetesCorticiales Corticiaceae Phanerochaete sp Agaricomycetes PolyporalesCoriolaceae Agaricomycetes Polyporales Coriolaceae Trametes sp.Agaricomycetes Polyporales Coriolaceae Trametes hirsuta AgaricomycetesPolyporales Coriolaceae Trametes villosa Agaricomycetes RussulalesPeniophoraceae Microbotryomycetes Sporidiobolales MicrobotryomycetesSporidiobolales Sporidiobolaceae Rhodotorula sp. MicrobotryomycetesSporidiobolales Sporidiobolaceae Rhodotorula mucilaginosaTremellomycetes Tremellomycetes Tremellales Tremellomycetes TremellalesTremellaceae Cryptococcus sp Tremellomycetes Tremellales TremellaceaeCryptococcus skinneri Tremellomycetes Tremellales Tremellaceae Tremellasp.

Example 14 Fungal Endophyte Seed Treatment Leads to Modulation ofPhytohormone Levels in Plants Grown from the Seed

To determine whether fungal endophyte seed treatment affectsphytohormone levels in plants grown from the seed, tissue was harvestedfrom the root or third true leaf of cotton plants inoculated with eitherendophytic Beauveria bassiana or Paecilomyces lilacinus. The experimentwas done with three endophyte treatments (uncolonized control, B.bassiana or P. lilacinus) and, for Beauveria bassiana, two herbivorytreatments (no aphids, or aphid herbivory for either 1, 4, 8, 24 or 48hours). Phytohormone levels for abscisic acid (ABA), tuberonic acid(12-OH-JA, an oxidation product of JA-Ile) (TA), ascorbic acid (AA),12-Oxophytodienoic acid (a JA precursor) (OPDA), JA isoleucine (JA-Ile),and salicylic acid (SA) were assessed by LC-MS in leaf and root tissuesseparately. All phytohormone level comparisons were made versus plantsin the uncolonized control group with significance at P<0.05.Phytohormone levels in plants grown from seed treated with Beauveriabassiana are shown in Table 3, and phytohormone levels in plants grownfrom seed treated with Paecilomyces lilacinus are shown in Table 4.

TABLE 3 Phytohormone levels in plants grown from seed treated withBeauveria bassiana Herbivory Phytohormone TissueUpregulated/downregulated Tissue Upregulated/downregulated Yes ABALeaves Down at 8 hours of feeding Roots Upregulated at 48 hrs of feedingNo Not significant Upregulated Yes TA Leaves Not significant RootsUpreguated at 48 hrs of feeding No Not significant Not significant YesAA Leaves Down at 4 hrs up at 24 hrs Roots Up at 8 hrs down at 48 hrs NoNot significant Upregulated Yes OPDA Leaves Not significant Roots Up at4 hrs and 8 hrs No Not significant Upregulated Yes JA-Ile Leaves Up at48 hrs Roots Up at 48 hrs No Not significant Upregulated Yes SA LeavesUp at 1 hr, 8 hr, 24 and 48 hr Roots Down at 4 hr the rest n.s No Notsignificant Not significant

TABLE 4 Phytohormone levels in plants grown from seed treated withPaecilomyces lilacinus Yes ABA Leaves Down at 48 hrs Roots Up at 1 hrand 8 hrs Yes TA Leaves down at 4 and 8 hrs Roots up at 4 hrs Yes AALeaves down at 4 and 8 hrs Roots up at 4 hrs Yes OPDA Leaves down at 4and 8 hrs Roots Up at 4 and 48 hrs, down at 24 hrs Yes JA-Ile LeavesDown at 8 and 48 hrs Roots Up at 4 and 24 hrs Yes SA Leaves Up at 1 and4 hr, down at 8 hrs Roots Up at 1, down at 8 hrs

Example 15 Fungal Endophyte Seed Treatments Alter Traits in CertainCotton Cultivars in Field Trials

The 2014 field trials were executed in a similar fashion as described inExample 6. A field trial using isolates of listed below was conductedduring the summer. Each plot consisted of four 15.24 m (40 ft) rows,each separated by 101.6 cm (40 in), and there were 6 replicate plots pertreatment. Yield from plots treated with the described microbialcompositions was compared relative to the untreated control plots. Forthrips, this damage assessment was on a scale of 0-5; 0=no damage,1=noticeable feeding scars, but no stunting, 2=noticeable feeding and25% stunting, 3=feeding with blackened leaf terminals and 50% stunting,4=severe feeding and 75% stunting, and 5=severe feeding and 90%stunting. For fleahoppers, the number of insects per plant werequantified and reported as an average for each plot. FIG. 14 shows theyield improvement of crops when treated with the described microbialcompositions, for Delta Pine and Phytogen cultivars, respectively. FIG.15 shows the aggregated yield improvement of the microbes across the twocultivars. Bars represent 95% confidence intervals. FIG. 16A shows thebeneficial effect of 12 out of 15 microbial compositions tested on thripdamage in the Delta Pine cultivar. In the Phytogen cultivar, only 2 outof the 15 microbial compositions tested showed a benefit by reducingthrip damage. FIG. 16B shows the beneficial effect of reducingfleahopper damage in the Phytogen cultivar, where 6 out of the 15facultative fungal endophytes tested showed an average decrease infleahopper damage as compared to untreated cotton plants. In the DeltaPine cultivar, only one microbial composition showed a beneficial effecton fleahopper damage.

A number of other mid-season plant traits were also assessed in thefield to determine the effect of the described fungal endophytecompositions. FIG. 17A shows the beneficial increase of the describedmicrobial compositions on mid-season mean root length. FIG. 17B showsthe beneficial increase of the described fungal endophyte compositionson mid-season belowground weight. FIG. 18 shows the beneficial increaseof the described fungal endophyte compositions on mid-season canopytemperature for both Delta Pine and Phyton cultivars. FIG. 19 shows thebeneficial increase of the described fungal endophyte compositions onmid-season NDVI (Normalized Difference Vegetation Index) for both DeltaPine and Phytogen cultivars. NDVI is a measure of chlorophyll content.FIG. 20 shows the beneficial increase of the described fungal endophytecompositions on mid-season first-position square retention for bothDelta Pine and Phytogen cultivars. FIG. 21 and FIG. 22 show themodulation (up in July and down in August) of mid-season plant heightwhen treated with the described fungal endophyte compositions for bothDelta Pine and Phytogen cultivars. FIG. 23 shows increased biomass inthe plants treated with endophytes (right half of the image) compared tountreated control (left half of the image).

In FIGS. 15 through 22, TAM505 is Acremonium sp., TAM32 is Epicoccumnigrum, TAM534 is Cladosporium urdinicola, TAM244 is Cladosporium sp.,TAM514 is Cladosporium urdinicola, TAM474 is Cladosporiumcladosporoides, TAM554 is Chaetomium globosum, TAM15 is Exserohilum sp.,TAM488 is Epicoccum nigrum, TAM452 is Cladosporium urdinicola, TAM490 isPaecilomyces lilacinus, TAMBB is Beauveria bassiana, TAM105 isCochliobolus sp., TAM189 is Bipolaris sp., and TAM47 is Epicoccumnigrum.

Example 16 Fungal Endophyte Seed Treatments Provide Drought Tolerance inCotton Cultivars in Greenhouse Trials

Cotton plants were germinated from endophyte-treated and untreatedcontrol seeds in the greenhouse. All seeds watered for 7 days or untilcotyledon stage using pre-determined soil saturation volume of water perplant. At 7 DAP, water was withheld from water stressed plants whilecontrols continued to be watered. Time to wilt and time to death weremeasured at a max of 21 DAP. The data in FIG. 24 shows the mean time towilt, and the data in FIG. 25 shows the mean time to death. Endophytetreatment increased the survival of plants subjected to drought stressin both the Delta Pine (DTP) and the Phytogen (PHY) cultivars. In FIGS.24 and 25, endophyte number 194 is Epicoccum nigrum, 249 is Cladosporiumcladosporioides, 355 is Chaetomium globusum, 46 is Epicoccum sp., 463 isCladosporium sp., 534 is Cladosporium uredinicola, 554 is Chaetomiumglobosum, 58 is Epicoccum nigrum, and control is no endophyte treatment.

Example 17 Identification of Fungal Endophytes with at Least 97%Identity to Those in Table 1

All known fungal endophytes with 97% identity to SEQ ID NO:7 through SEQID NO:77 were identified and are listed here by accession number:FJ425672, AY526296, JQ760047, UDB014465, KC662098, HQ649874, JQ764783,EU881906, KF251285, JQ862870, AB019364, AB594796, JF773666, JN034678,KC343142, EU707899, AB627855, GU138704, JN695549, DQ279491, HM776417,AB361643, DQ782839, AF222826, EU682199, DQ782833, EU054429, FJ025275,AY354239, AF222828, GU721921, GU721920, DQ093715, AJ309335, FR774125,JQ747741, EF042603, KC968942, HE584924, AY740158, FJ645268, HQ692590,GQ203786, AY233867, HE579398, AB777497, KF435523, DQ420778, JQ649365,AJ271430, GQ996183, EF070423, FJ172277, AF483612, JX675127, EF070420,EF070421, AB741597, JN225408, DQ019364, KF251279, EF194151, EU977196,JX981477, EU686115, JX021531, FJ527863, AJ302451, AJ302455, JN975370,EU754952, AF284388, KF296855, AF502785, JX317207, AF502781, DQ278915,EU686867, KC179120, HM991270, AF284384, DQ632670, JQ759806, JQ747685,EU885302, GU721781, EF434047, EF505854, JQ666587, JQ619887, GQ919270,KF531831, AB627854, DQ914679, DQ914681, Q599592, DQ279490, DQ660336,JX069862, AB607957, HE820869, FJ859345, JX966567, GU910230, AB627850,JX144030, DQ914723, HM595556, KC771473, DQ849310, EU179868, KF312152,JN890447, JX042854, EU554174, JN198518, HM992813, JQ845947, KF251310,JQ758707, AM930536, KF296912, JN865204, JN943512, GQ921743, EU245000,EU977304, EU144787, HE579322, HE579402, GU910171, HE792919, KC960885,DQ485941, JN604449, HQ607913, AF502620, DQ468027, JX944132, JN207338,JQ922240, JN207336, JX559559, JN207330, JN207333, HE820882, JX969625,HQ339994, JF744950, HE584937, JN120351, JX298885, DQ872671, AJ877102,JQ081564, DQ019391, AF071342, EF104180, JQ759755, GU827492, JN418769,GU324757, JX984750, JX256420, KF436271, JX205162, JN712450, KF435911,GU367905, JX416919, KC315933, JQ736648, AY904051, AF404126, J466722,HE584965, JN890282, HE584966, HQ166312, KC305124, HE977536, KC305128,AY907040, JF710504, AF483609, AJ302460, AJ302461, AJ302462, AY969615,EU685981, 75615, AJ302468, FJ210503, GU237860, JX960591, JX143632,HM044649, EU164404, HE584824, HQ116406, DQ156342, JX416911, U75617,GU721359, KC427041, EU254839, JX262800, KC179307, HQ107993, KF361474,GU721420, HM053659, EF619702, EU686156, HE820839, HQ634617, GU721810,AB277211, AJ302417, KC315945, JQ002571, AM237457, AF009805, JX489795,EU680554, KC507199, FJ236723, HQ692618, JN846717, JX944160, JQ585672,KF435573, EU520590, HM581946, DQ250382, JX243908, KC343184, KC485454,GQ479695, GU237760, KF147147, EF619849, GU237767, GU237766, AB818997,AF502847, EU683672, KF225801, KC965743, AJ488254, DQ825983, JNO31007,DQ825985, KF028765, AB818999, HQ238268, EU685984, KC966180, HE998711,HQ533007, AM113729, KF251637, FN394692, KF435172, JN207307, JQ814305,HM770988, KC145175, AB511813, EU552102, AJ309344, EU645686, JQ936328,JNO38492, DQ875349, EU977228, JQ814357, KF040480, JX317350, DQ401548,DQ318195, DQ318194, GU721776, KF193449, AF178544, AM262354, AB540567,AY627787, HE792907, HE579333, EU445372, AF362069, GU973687, HM053663,AB374284, DQ062977, GU237797, JQ760783, EF029240, HM751829, FM200445,AY953383, AY233922, JF742784, HM626650, FN610871, JX155902, JX006065,AB566289, AF163078, AY344976, AB566287, AF282089, AY251441, AF395693,JQ761899, AJ315835, HQ187633, KC287233, AJ315831, HE820745, JN418779,M13906, JQ761896, AJ315838, AY536373, HQ328035, JX838793, JQ758986,HQ166357, JN163855, KC965595, JN545789, JN545788, GU944558, HE579247,KF296900, EF377335, KC965954, GU269703, AB095511, EF419913, DQ993641,AB325678, HQ223035, AY513945, FJ197013, FR799277, HM071900, JN207293,FJ025268, JQ758966, GU138733, GU138730, DQ267595, GQ919269, JF770450,GU138734, DQ279488, DQ279486, DQ242472, EU164804, EF104177, GU366726,KF212243, DQ923534, GU079598, JX987761, JX984765, AY585343, JQ769260,GU721919, DQ923538, EU686756, EU040222, U75626, GU004264, EU686753,JQ765651, JX270629, JN943408, EF042604, AJ271588, HE579386, GQ479556,JQ759962, JX317413, EU516867, DQ780361, JQ905644, HQ649792, JQ247355,FN386296, AY004778, DQ102374, KF251383, GU237835, DQ383642, FN868479,GU237814, KC343032, JN943394, HQ450001, KC800573, AB217793, GQ851883,EU330630, JF309198, AY489281, GU325687, JX399008, AB164703, EF159407,AJ302429, UDB008141, UDB008140, FM200496, AJ302426, AJ302422, AJ302423,JQ683725, KF193481, JQ683727, HE792931, AB220252, FJ013057, DQ286207,JQ759811, JF414842, JX088707, JN415754, AY787715, JX559577, KC776206,GU166440, KC460867, FJ515595, KF056850, DQ118964, KC806227, KC631802,EU823315, AY528970, HQ116401, JX317516, KF251313, KC800565, AF502705,AF502810, M747697, AY527407, EU680518, AJ621773, AB374285, HQ832827,GU174316, DQ974750, JN198507, JF749806, JQ782739, HQ023202, AY616234,KC965315, AB743781, EU554161, KC507201, HM036624, EF464164, JX391942,AB743995, FJ415474, AY647237, KC965503, AB540553, HQ377280, JX898571,JN969419, DQ166962, HM123519, GU237881, AB683953, AY681487, EU498738,EU687037, AB540550, EF394866, AY853245, EU680532, HQ450006, AM292674,KF435452, AF502638, JN890354, JX256427, JF773646, KC916704, FJ347031,JN572154, AF443850, AY273300, JQ247392, JQ247393, HQ316569, GU324760,AB120858, JF440978, HQ115719, JF440976, DQ124120, HQ022342, AF333138,AB255293, GQ999456, DQ286209, HE820785, AF451751, JNO38479, JQ044421,JQ044422, KC968911, KC492447, FM172902, AF437754, HM030631, HM595545,AY510424, JX414184, HQ184179, AB588822, JQ813816, JQ813817, FJ025255,AY745019, EU668292, HM216214, AF427105, EU479799, JQ769257, HM484866,EU301059, EU564808, AY265329, HQ701737, KC677889, AY907030, GU721349,AY304513, GU062277, AY907037, HM484859, AB576865, JX090109, UDB004179,JN692542, JQ327868, AY756490, JN890185, JX042994, FJ613832, AF009815,HQ332534, AF009816, EU686781, DQ520639, KC247154, HE820841, HE820847,JN717228, JX944174, GU721348, AB444657, KF435560, JQ585546, JQ775577,UDB004443, JF744968, KF192823, JN102440, AM504058, JX164074, GU907781,HQ889707, FJ612980, KF251355, AF502854, AF350291, HQ649989, GU966521,FJ481149, AY916491, AB444663, FR799197, KC691458, HE820786, JN802324,AF149926, AY372686, AY233908, HQ631033, UDB004677, KF251596, EU479757,GU079602, KC691456, DQ420883, DQ914680, DQ914683, KC305134, JN207313,AB512307, JN807326, GQ395365, JN207256, FJ425678, AB000932, JN207252,KF293814, GU138728, AY160210, UDB015006, KC565735, FJ524302, AF404127,EU272486, JF796251, JF439458, AY304511, KC592278, JX143583, JF440977,EU686925, JX982370, EU687082, JX966607, GU222370, JN687988, JN006771,JX436806, JQ936201, KF481950, AF178551, KC181937, JX144778, DQ790541,JF796076, JX898576, JX418352, AF097902, FJ411320, AF309617, FR863589,HM469970, AF163069, KF582795, AB566293, HE820790, GQ267191, JX130356,JN049828, HM060596, KF436001, GQ919283, HQ832834, JN049822, EU041786,AB594789, HE579259, HE584944, GU004268, GU237770, GQ921765, HE579253,KC305158, AF043599, GQ267190, AY344968, JN601031, JN969420, GU328624,AB540507, AM691002, JN102384, EU480019, JN545815, DQ993651, JX130360,JX398990, AY969704, KF251559, AF395695, HQ449993, U94714, KF435968,JX966550, AB859762, JF749808, U94713, GU981750, AF177152, FJ430599,JB647433, GU981756, GU981757, EF104164, JN802311, GQ266146, HQ445083,JX155909, KF436256, DQ318204, GU078649, JN890115, DQ386141, GQ999487,EU686744, FJ426983, UDB013022, FN435799, EF600976, HM596012, JF825143,AM711381, EU816668, AJ972833, JQ905735, AF004686, EU266103, EU266107,HQ166334, EF679384, UDB004580, AM691001, JX399012, KC460880, JX982437,AB482221, AM292048, KF251253, AF350308, JF502446, JQ905803, KC179320,KF251393, GU053815, DQ323686, DQ323681, KC343119, HE820747, KF251529,DQ676536, U17215, DQ278919, EU489950, FR668016, GU903287, AJ302439,AJ302438, AJ302435, AJ302434, JN807325, AB741584, KC790941, DQ394387,FJ403513, GQ461566, KF193491, KC305164, AF502895, GU237707, EU977520,AB247177, AB482220, AY929321, GU004278, AB247171, GU461294, GU461295,JX123570, AY684241, EU686968, JX944143, JN871718, JQ796813, HQ829122,KF435590, KC806231, JX414183, GU944858, AF502733, JN662314, HQ022970,AY510418, KC623569, KC216145, KF129059, DQ279515, KF251526, JN192379,JN192376, HM140630, DQ006928, AF011289, EU089663, FJ825373, DQ307292,JN890424, KF155521, AB670714, GQ927271, AB670717, B670711, AB670713,KF435279, GU053814, KF435375, JX414188, AF033422, GU225946, EU520610,JF773645, KC595884, KC965570, DQ812921, EU885299, DQ078757, FJ612618,KF018920, JX077035, EU686911, JX270567, HE579352, EU885297, FJ418185,DQ914724, HQ608112, HQ450016, GU174399, JN890327, HM999913, GU079580,HE584936, JQ765675, GU726947, JQ765670, HM588120, AY969986, JN120335,JQ247384, HQ891112, JQ769297, JN207242, EU002888, EU479803, AY365468,AF163083, DQ534482, KC146356, KF436052, AF416460, JX537970, JX156018,AY907035, GQ241278, AF409972, JQ388941, FR668022, EU687151, DQ468026,AY251418, AB508842, AB508840, DQ233665, GU721949, AJ302444, JF927155,AJ302442, GU721976, AJ302440, KC790931, UDB004433, GU328539, EU479791,HQ649905, KC797566, JQ753968, GU721449, HQ701742, AY613410, GU062246,AY907045, HM991267, DQ979608, JQ781840, GU721442, EU426553, DQ980024,HQ634638, AF222836, GU222372, AY969338, EF104158, AY431101, JQ081415,FJ649318, AY152583, JN943058, EU885294, HQ231255, FJ179477, EU304350,KC005785, FR799224, EF070422, HQ533789, AJ289870, KF025952, HQ611347,DQ485934, KC989106, JQ081921, HE820871, AF404125, JN603182, KF436170,HQ832964, DQ185074, KC216108, JN102460, GU553324, DQ318207, HQ589260,AB819001, AY699669, EU812501, AB819004, HQ436065, KC013976, KF251204,KF435307, AF249905, EF029217, EF029216, FJ708614, EF029198, JQ517314,GU199416, HM180398, EU479748, GU721599, DQ185081, EF104175, JX021528,KF251430, AY611071, AY329221, JN207241, HM235963, JN890375, JF506092,KF193461, KF453551, HM123501, HM051074, AB255269, HQ904082, KF193500,FN562038, GU721911, EF417805, KF193504, KF028766, HE579312, EF433991,KF144910, KF144911, FM200450, AF163090, AB444665, AB444664, HQ649964,AB444666, AB444661, AY528998, DQ525492, KC870889, EF543844, GU073125,AY684240, JN163853, EU680538, AF395694, KC179102, KC778197, JN102425,DQ520638, EU244997, GU994552, DQ279527, KC179418, EF495164, AY999117,JX860441, JQ793663, DQ836775, EU479964, AY772736, AJ875343, KC013972,AJ875346, AY208785, HE614864, HF570009, KF435344, KC148376, EF641857,JX625368, AB512308, KC305146, AY266384, KC662096, HE579269, GU004277,GU004276, EF504668, EU687114, GU004272, GU004271, EU516731, KC213751,JN102394, HQ654776, JQ862729, EU687052, JX868653, FJ172294, JX130355,HE584891, FJ427063, GQ996174, FN252438, AJ633598, JX398987, EU245009,HM069466, FJ859344, JN942165, FJ785433, EF504592, HQ449989, HQ449988,JN120346, JX868648, EF600969, HQ529711, JN383815, KF003112, JN890192,GU981748, EU715654, EF535663, GU328634, UDB004320, GQ999475, FR731421,GU322457, EF550969, GU322450, FJ477838, KC305130, AJ247519, JQ026214,AJ972825, KC305135, EU520614, EU338415, JQ747670, EU040241, HE584979,KF477240, HM162095, AB746179, KC963934, AY906949, JN975339, EU520120,HM071902, JX399005, EU828350, JX399006, EF070418, FJ025231, EF070415,JN859327, JQ517292, JX399009, KF297004, JN618372, AY233888, EU784271,AM292673, EU514295, GQ921804, GU595027, HM008727, GU174426, JN673038,AF442801, EU686126, JF440982, EU754960, GQ154505, GU055711, FJ175159,KC354573, DQ993639, JQ621881, JN102454, AY177233, FJ013071, AY566992,GQ120971, EF408555, JX317505, AF524905, FJ887922, AF264905, AF264906,HM997113, EF619857, KC537805, KC537804, FJ887928, AB255303, HQ671302,FJ210537, FN386267, HQ649813, GU083033, KF251334, GU721297, KC181926,DQ832329, JQ781696, KF251233, KF251234, GQ505688, AJ437294, AJ437295,EF679363, HE820831, FN868450, GU174305, AY428866, AY956759, JQ759940,DQ489291, AJ271418, AY157952, EU784408, FJ427055, EF419900, FN813731,FJ427059, KF435462, JQ860113, KF209290, JF439437, KC565714, FJ228189,AF377282, JQ814364, HM991266, EF458676, AY762046, JN048884, HQ896484,HE579345, AB444659, EU076958, HQ402674, AF540504, AM922204, EU479758,JN943840, JN943841, FJ427025, KC584194, AF502754, FJ418192, KC343004,AB524806, AJ877224, DQ394377, FJ427028, AF282090, GQ927270, EU178738,DQ059579, EF535699, KF040479, AF163085, JX256429, AY999125, KF477238,KC513506, GQ999534, GU237837, EU002898, HM164732, AF443193, AJ315828,AJ315829, AY586560, JX868722, EU686847, DQ875350, DQ421277, AM176740,JX280875, AM691003, KF302463, GQ921786, KC965801, AM691004, EF452446,EU040235, KC662103, KC662102, AY251073, DQ993637, AY489282, FJ151434,JQ936199, EF505495, JN163856, JN659510, EF452449, EF504607.1,GQ516009.1, GQ508761.1, KC8008470.1, JX187590.1, GQ508832.1, KC800841.1,KC800840.1, EF504876.1, HQ540685.1, EF505180.1, AY842353.1, GU014821.1,FJ761203.1, GQ510033.1, EF504642.1, GU014822.1, AY998786.1, AB581046.1,EF452470.1, FJ907534.1, EF504721.1, Y08744.2, FJ757587.1, GU014820.1,AF400896.1, KC800831.1, EF505804.1, EF505121.1, JX187587.1, KC800858.1,GQ866210.1, GQ522120.1, Y10748.1, EF504853.1, EF452471.1, KJ834329.1,AB581446.1, JX187588.1, AF163061.1, AB632670.1, Y08746.1, EF505082.1,JX187589.1, EF504723.1, AF400889.1, KC800835.1, and EF505282.1.

Example 18 Endophytes and Combination Thereof

The protocols as described in Examples 1-16 are used in connection withthe endophytes of Table 1 to confirm beneficial properties on planthealth, such as yield and/or past resistance, for example. Inparticular, endophytes from Table 1 are employed in a syntheticcombination with a plant as described herein with crop plants, such ascotton. Any single or combination of endophytes listed in Table 1 canalso be used in this manner, employing for example seed coatings orfoliar, soil, or rhizosphere applications. A seed composition maycomprise seeds and any combination of endophytes listed in Table 1.Endophytes listed in Table 1 or combinations thereof are thus employedin methods for preventing pest infestation, increased yield, treating apest infestation, manufacturing pest-resistant seeds; or increasing ayield or reducing loss of a crop according to the methods of Examples1-15.

1.-20. (canceled)
 21. A method for improving drought resistance in acotton plant, the method comprising: contacting a cotton plant or a seedof said cotton plant with a formulation comprising purified filamentous,spore-forming, facultative fungal endophytes of at least one species,wherein the facultative fungal endophytes are present in the formulationin an amount effective to improve drought resistance of the cotton plantor a cotton plant grown from the seed compared to a reference cottonplant or a cotton plant grown from a reference seed.
 22. The method ofclaim 21, wherein the formulation contains at least 100 (10̂2) spores/mlor 100,000 (10̂5) spores/g dry weight of the facultative fungalendophytes.
 23. The method of claim 21, wherein the facultative fungalendophytes are present in the formulation in an amount effective toincrease biomass. 24.-26. (canceled)
 27. The method of claim 21, whereinthe facultative fungal endophytes are present in the formulation in anamount effective to reduce yield loss.
 28. The method of claim 27,wherein yield loss is reduced by at least 5%.
 29. The method of claim27, wherein the reduction in yield loss results in a yield increase ofat least 5%.
 30. The method of claim 21, wherein enhanced resistance todrought stress is assessed by withholding water from 7-day old seedlingsof the cotton plant grown in the greenhouse, wherein the seedlings haveincreased time to wilt as compared to a seedling grown from a referenceseed.
 31. A synthetic combination of a cotton seed and purifiedfilamentous, spore-forming, facultative fungal endophytes of at leastone species, wherein the facultative fungal endophytes are present inthe formulation in an amount effective to improve drought resistance ofthe cotton plant grown from the seed compared to a plant grown from areference seed.
 32. The synthetic combination of claim 31, wherein thefacultative fungal endophytes are present at a concentration of at least100 (10̂2) spores/seed on the surface of the seed.
 33. The syntheticcombination of claim 31, wherein the facultative fungal endophytes arepresent in the formulation in an amount effective to increase biomass.34.-36. (canceled)
 37. The synthetic combination of claim 31, whereinthe facultative fungal endophytes are present in the formulation in anamount effective to reduce yield loss.
 38. The synthetic combination ofclaim 37, wherein yield loss is reduced by at least 5%.
 39. Thesynthetic combination of claim 37, wherein the reduction in yield lossresults in a yield increase of at least 5%.
 40. The syntheticcombination of claim 31, wherein enhanced resistance to drought stressis assessed by withholding water from 7-day old seedlings of the cottonplant grown in the greenhouse, wherein the seedlings have increased timeto wilt as compared to a seedling grown from a reference seed.